TWI221855B - Process for synthesizing nucleic acid - Google Patents

Abstract

The present invention relates to an oligonucleotide having a novel structure and a method of synthesizing nucleic acid by using the same as a primer. This oligonucleotide is provided at the 5'-side of the primer with a nucleotide sequence substantially the same as a region synthesized with this primer as the origin of synthesis. The present invention realizes synthesis of nucleic acid based on an isothermal reaction with a simple constitution of reagents. Further, the present invention provides a method of synthesizing highly specific nucleic acid on the basis of this method of synthesizing nucleic acid.

Description

五、發明說明（l) 技術領域 本發明是有關於作為以核 鹽基序列構成之核酸的合成 方法^有用的特定 背景拮術 傳特=為基礎的分析方法，是對遺 傳疾患、癌化、微生物的識別等因J是:b法對於遺 此外’基因作為檢出對象之時，利的手段。 類，耗時又耗力之操作過程的情形。如培養之 τ 5兄如此’存在試料中之目的基因量稀少的情 ::疋不太容易檢出標的基因’戶斤以增幅標的基‘： =訊息等步驟就變成必要的條件。較為人熟知 曰田私的基因之方法為聚合酶鏈鎖反應（pCR) (Sciewe， 230’ 1350-1354，1 985 ) 〇PCR法是現在對於活體外（in’ \itro)增幅核酸之技術最為常用的方法。此方法乃基於以 指數增幅的效果’是具有高敏感度且優良的檢出方法。而 且’因為增幅生成物之DM可以回收，可以將之廣泛應用 於基因選殖（cloning)及構造決定等基因工程之重要的工 具。然而，對於PCR法而言，為實驗目的必須備有特別的 溫度調節裝置；而因為增幅反應是以指數倍增，故定量性 的問靖也不得不考慮；試料及反應液若是受到外部污染， 誤混入的核酸也可能成為增幅反應的模板，容易受到污染 而影響結果等也是常為人所話病的問題。 伴隨著基因體情報的累積，單一核苷酸多型性（SNPs;V. Description of the invention (l) TECHNICAL FIELD The present invention relates to a method for synthesizing nucleic acids composed of nuclear salt-based sequences. ^ Useful specific background analysis method based on antagonism and passivity = is an analysis method for genetic diseases, cancer, The reason for the identification of microorganisms is J. The b method is a useful method when the gene is detected. A situation that is time-consuming and labor-intensive. For example, when the τ 5 brother is cultivated, the target gene is scarce in the sample: :: 疋 It is not easy to detect the target gene 'To increase the target base': = Information and other steps become necessary conditions. The more well-known method of Tian Tian's gene is the polymerase chain reaction (pCR) (Sciewe, 230 '1350-1354, 1 985). PCR method is currently the most popular technique for amplifying nucleic acids in vitro (in' \ itro). commonly used ways. This method is based on the effect of an exponential increase ', which is a highly sensitive and excellent detection method. And because the DM of the amplified product can be recovered, it can be widely used as an important tool for genetic engineering such as gene cloning and structural determination. However, for the PCR method, a special temperature adjustment device must be provided for the purpose of the experiment; and because the amplification reaction is an exponential multiplication, quantitative questions must also be considered; if the sample and the reaction solution are subject to external contamination, errors may occur. The mixed nucleic acid may also become a template for the amplification reaction, which is easily affected by contamination and affects the results. With the accumulation of genomic information, single nucleotide polymorphisms (SNPs;

第4頁 1221855 五、發明說明（2) single nucleotide polymorphism)的分析也成為注目的 焦點。引子（primer)的鹽基序列中含有SNPs之設計，可以 利用PCR以檢出SNPs。也就是說，依反應生成物之存否可 得知是否存在引子互補的鹽基序列。然而對於PCr而言， 萬一發生錯誤的互補鍵合成反應時，就會變成生成物之後 的反應皆是以錯誤的模板進行而得到錯誤結果的情形。現 貝中’引子的末端假使僅有1個鹽基不同，將不容易對PCR 作嚴密控制。依據於此，利用pCR進行SNps的檢出有改善 其特異性的必要。 ° 另一方面，以接合酶（ligase)為基礎的核酸合成方法 也已經被實用化。LCR 法（Ligase chain Reaction，Page 4 1221855 V. Description of the invention (2) The analysis of single nucleotide polymorphism has also become the focus of attention. The design of the primer contains the SNPs in the base sequence. PCR can be used to detect the SNPs. In other words, it can be known whether there is a base sequence complementary to the primers depending on the existence of the reaction product. However, in the case of PCr, if an erroneous complementary bond synthesis reaction occurs, the reaction after the product will be performed with the wrong template and the wrong result will be obtained. If only one base is different in the terminus of the primer of the present primer, it will not be easy to tightly control the PCR. Based on this, detection of SNps by pCR is necessary to improve its specificity. ° On the other hand, nucleic acid synthesis methods based on ligase have also been put into practical use. LCR method (Ligase chain Reaction,

Laffler TG； Carrino JJ； Marshall RL; Ann. Biol.Laffler TG; Carrino JJ; Marshall RL; Ann. Biol.

Cl in· (Paris)，i 993，51 : 9，"I -6)的基本原理是對檢 出對象序列上鄰接的兩個探針（probe)進行雜交 (hybndize)，以接合酶將兩者連接之反應。當標的鹽基 序列不存在的情形下，此二探針就不能連結，所以連結生 f物之存在就成為標的鹽基序列的指標。LCR法也必須有 口成後互補鏈與模板分離的溫度控制條件，因此也伴有盥 法相同的問題。LCR法也有報告指出，在鄰接探針的/中 复 、二序、（gap)，再加以DNA聚合酶（DNA polymerase)將 Z充填的工程，以改善其特異性。可是，此種改良方 纲快I的確可以達到改良特異性的問題’仍然留下了溫度 1 =:問題沒有解決。此外，因為增加了必要的酵素，成 本上也作了犧牲。The basic principle of Cl in · (Paris), i 993, 51: 9, " I -6) is to hybridize (hybndize) two probes adjacent to each other in the detection target sequence, and use ligase to hybridize the two probes. The response of the connection. When the target base sequence does not exist, the two probes cannot be linked, so the existence of the linking bio-product becomes an indicator of the target base sequence. The LCR method must also have temperature control conditions for the separation of the complementary strand from the template after oral completion, and therefore also has the same problems as the wash method. The LCR method has also been reported that the process of filling the Z with adjacent probes, secondary sequences, and DNA polymerase (DNA polymerase) to improve the specificity. However, such an improvement can quickly achieve the problem of improvement specificity ', and it still leaves a temperature of 1 =: the problem is not solved. In addition, because necessary enzymes are added, costs are sacrificed.

1221855 五、發明說明（3) 以檢出對象作為模板，利用含有互補序列⑽A進行增 幅的方法中，名為SDA(Strand Displacement1221855 V. Description of the invention (3) The method using the detected object as a template and using the complementary sequence ⑽A for amplification is called SDA (Strand Displacement

Amplification) 〔Proc· Natl. Acad· Sci. USA 89 392-396; 1992〕 〔Nucleic Acid· Res·， 20 1 69 1 - 1 696; 1 992〕的方法也為人所熟知。SDA法就是將某 鹽基序列的3’端互補的引子作為合成起點，進行互補鏈合 成反應，而在5’端兩股鏈的領域進行鏈置換反應的同時了 利用特殊的DNA聚合酶進行互補鏈合成反應的方"法。且在 以下本說明書中，單以5,端、或是3,端表示時，/代表哪一 方作為模板和鏈的關係。5’端的兩股鏈部份被新合成互補 鏈置換（displacement)，故名之為SDA法。SDA法中，引子 預先***了限制酶認識序歹，而實現 :二=須的溫度變化工程。也就是說，以限 制酶&成的缺口（nick)，提供3’_〇h基作為互補鏈合 此處開始進行鏈置換合成反應，將先合成之 互補鏈中的一股游離，再將之作為模板利用於接下來的互 補鍵合成反應。這籍S D A法廡》γ p p p、^ 調控㈣。&種SDA法廢除了 PCR法所必須的複雜溫度 1而，SDA法中加入了鏈置換型的Ma 組合限制酶以進行反應。增加必要= 兩r鏈，❿：：的因素。此外’所採用的限制酶並非切斷 兩版鏈而疋為了導入缺口（即是只切斷一股）， 股=須具有酵素分解财性，所以在 不利用硫代，TP之類的dNTp誘導物作為基質初=付Amplification) [Proc. Natl. Acad. Sci. USA 89 392-396; 1992] [Nucleic Acid. Res., 20 1 69 1-1 696; 1 992] is also well known. The SDA method is to use a complementary primer at the 3 'end of a certain base sequence as a starting point for the synthesis of a complementary strand, and perform a strand displacement reaction in the area of two strands at the 5' end while using a special DNA polymerase to complement Method of chain synthesis reaction. And in the following description, when it is represented by 5, or 3, only, / represents which one is used as the template and chain relationship. The 5 'end of the two strands is replaced by a newly synthesized complementary strand, hence the name SDA method. In the SDA method, the primers are pre-inserted with the restriction enzyme recognition sequence, and the realization is as follows: Two = the temperature change engineering of the whisker. In other words, the restriction enzyme & nick provides a 3'_〇h group as a complementary strand. Here, a strand displacement synthesis reaction is started. It is used as a template for the subsequent complementary bond synthesis reaction. This book S D A method "γ p p p, ^ regulation ㈣. A kind of SDA method abolishes the complicated temperature necessary for the PCR method 1 and a chain displacement type Ma combination restriction enzyme is added to the SDA method to perform the reaction. Addition necessary = two r chains, ❿ :: factor. In addition, the restriction enzyme used is not to cut the two versions of the chain, but in order to introduce a gap (that is, to cut only one strand), the strand must have the property of enzyme decomposition, so dNTp induction such as TP is not used. Substance as matrix

第6頁 1221855Page 6 1221855

以SDA法增巾昌之產物就變成與天然核 文到了限制。此外也伴隨著花費增加 ς :τ就 跑應用於未知_，不㈣⑽合二。而且，’將 著與為了導入缺口所使用之限制酶 s 子在 列之可能性。♦人擔心這樣子二相同的鹽基序 成反應之反應完全性。+的狀况將可能影響互補鏈合 不需要複雜之溫度調控機制的核酸增幅方法 NASBA (以核酸序列為基礎的增幅，亦稱 ’ 節的增幅）為人所知。NASBA是將標的RNA作為模板， 加有T7啟動子（n pr〇m〇ter)的探針，利用dna聚合酶進行 DNA合成，再以第二個探針合成雙股鏈，以生成的雙股⑽八 鏈作為模板，再用T7 RNA聚合酶進行轉錄，使用增幅大量 RNA 的反應系統（Nature，35〇，91一92， 1991)。^別八在雙 股DNA鏈完成之前仍有幾個階段的加熱變性工程要求，但 是之後的T7 RNA聚合酶所進行的轉錄工程就可以在恆溫下 進行。然而，因為反應必須組合反轉錄酶、RNaseii、dna 聚合酶及T7 RNA聚合酶等多種酵素，與SDA —樣，具有花 費較貴的缺點。此外，為使多種酵素進行反應，條件設定 也變得較困難，要作為一般普及化的分析方法也不太容 易。所以，在已知的核酸增幅反應中，諸如複雜的溫度調 控問題、或是需要多種酵素參與反應等缺陷仍存在。 還有，這些已知的核酸合成反應，不只是犧牲掉特異 性與經費上的耗損，也完全沒有對於核酸合成反應是否更The use of SDA method to increase the product of Changchang has become a limitation with natural verification. In addition, with the increase in cost ς: τ will be applied to the unknown _, not both. Moreover, it will be listed with the restriction enzyme s which is used to introduce the gap. ♦ People are worried about the completeness of the reaction of the same salt motif. The state of + will likely affect complementary strands. A nucleic acid amplification method that does not require a complex temperature regulation mechanism, NASBA (Amplification Based on Nucleic Acid Sequences, also known as ‘Section Amplification’) is known. NASBA uses the target RNA as a template, a probe with a T7 promoter (n promote), DNA synthesis using dna polymerase, and a double strand with a second probe to generate the double strand. The eight strands were used as a template, and then T7 RNA polymerase was used for transcription, using a reaction system that amplified a large amount of RNA (Nature, 35, 91-92, 1991). ^ Beijing Ba still needs several stages of thermal denaturation engineering before the completion of the double-stranded DNA strand, but the subsequent T7 RNA polymerase transcription engineering can be performed at a constant temperature. However, because the reaction must combine various enzymes such as reverse transcriptase, RNaseii, dna polymerase, and T7 RNA polymerase, like SDA, it has the disadvantage of being expensive. In addition, it is difficult to set conditions for the reaction of various enzymes, and it is not easy to be used as a general analytical method. Therefore, in the known nucleic acid amplification reactions, defects such as complex temperature control problems or the need for multiple enzymes to participate in the reaction still exist. In addition, these known nucleic acid synthesis reactions not only sacrifice specificity and financial losses, but also do not at all whether they are more sensitive to nucleic acid synthesis reactions.

1221855 五、發明說明（5) 有效率的試驗報告。例如，名為RCA (Rolling-circle amplificati〇n)的方法是在標的鹽基序列存在下，與閉鎖 (padlock)探針互補的鹽基序列可以連續合成一股dNa鏈 (Paul M. Lizardi et al., Nature Genetics 19, 225-232，July，1 998) qRCA 是一股寡核苷酸 (oligonucleotide)的5’末端及3’末端在LCR，構成鄰接探 針特殊的閉鎖構造，而利用此構造進行反應。接著配合利 用作為鏈置換型互補鏈合成反應觸酶的聚合酶，在標的鹽 基序列存在的情形下，使之接合（ligati〇n)環化而成的閉 鎖探針作為模板，啟動（trigger )連續的互補鏈合成反 應。在同樣鹽基序列的領域中連續進行反應而生成一股連 續出現相同構造的核酸鏈。對於此一股核酸鏈再進一步地 加入引子與之結合，進行互補鏈合成反應，而實現高度增 幅的目的。然而，此方法依然殘留有需要多種酵素的缺曰 點。此外，互補鏈合成反應的啟動是依存著2個鄰接領域 的連結反應，其特異性在原理上與LCR是同等的程产 對於所謂供給3，-0H而言，在3,端具有同一鏈:鹽基 序列互補的序列，已知有利用在末端形成髮夾狀環 土 (hairpin loop)的方法而提供3，—〇H (Gene，71，^ 1 988)。由這種髮夾狀環開始，以本身作為模板進補 鏈合成反應，而生成互補鹽基序列構成的一股 如PCT/FR95/00891即是以互補鹽基序列連結的末ς ^二例 與同股鏈相結合的構造來實現反應的進行。然而刀、 須有在末端消除與互補鏈鹽基對的結合（b •法必 口 \uabe pa i r1ng)， A221855 五、發明說明（6) 而改構成同 伴隨著鹽基 微妙的平衡 的鹽基對結 衡狀態，與 鏈合成反應 率，必須考 術之初，設 I生成引子 列，增幅反 物。這是一 著進行的非 增幅反應效 除此之 月b作為換板 (EP713922) 也伴隨著形 性，使本方 且，不得不 領域所作的 此外對 (signal)增 每每利用伴 5-211873)。 一股鏈上之鹽基對結合的步驟（step)。此步驟 對結合之互補鹽基序列的末端，依存之而形成 關係以進行反應。也就是說，利用了與互補鏈 合及與同一股鏈上的鹽基對結合之間維持的平 同一股鏈上之鹽基序列結合之末端提供為互補 的起點。依據於此，為了達成高度的反應效 慮設定嚴密的反應條件。更進一步，採用此技 計的引子就要本身會形成環狀構造。因此，一 雙體（primer dimer)，不論有無標的鹽基序 應會自動開始進行，而形成非特異性的合成產 項嚴重的問題。而且，生成引子雙體後，伴隨 特異性合成反應會消耗掉引子，影響到目的物 率的低下。 外，對於DNA聚合酶，也有報告實現了利用不 的領域在同一股鏈上使3,端構造結合的試驗 此報告亦是利用末端部分的動態平衡，由於 成引子雙體而進行非特異性合成反應的可能、 $亦具有前述PCT/FR95/〇〇891相同的缺陷。而 = >主意針對不㉟&為DNA聚合酶模板之特 引子。 : = jNASBA的原理而延伸出的各種訊息 p芸:山〇 ，為了供給雙股鏈的啟動子領域， 通者末為髮夾狀構造的寡核苷酸（特開平 ^而這並非互補鏈合成3，-〇H之連續的供給。1221855 V. Description of invention (5) Efficient test report. For example, the method named RCA (Rolling-circle amplification) is to continuously synthesize a dNa chain in the presence of the target salt-based sequence, which is complementary to a padlock probe (Paul M. Lizardi et al. ., Nature Genetics 19, 225-232, July, 1 998) qRCA is a 5 'and 3' end of an oligonucleotide (oligonucleotide) at the LCR, which constitutes a special latching structure of the adjacent probe, and uses this structure Perform the reaction. Next, using a polymerase as a strand displacement type complementary strand synthesis reaction enzyme, in the presence of a target base sequence, a latching probe formed by ligating (ligation) cyclization as a template, and starting (trigger) Continuous complementary strand synthesis. The reaction is continuously performed in the same base sequence field to generate a nucleic acid chain having the same structure continuously. A primer is further added to this nucleic acid strand to combine with it, and a complementary strand synthesis reaction is performed to achieve the purpose of high amplification. However, this method still has the disadvantage of requiring multiple enzymes. In addition, the initiation of the complementary chain synthesis reaction is dependent on the connection reaction of two adjacent fields, and its specificity is theoretically equivalent to that of LCR. For the so-called supply 3, -0H, the same chain is present at the 3, end: A sequence complementary to the base sequence is known to provide 3, -OH (Gene, 71, ^ 988) by a method of forming a hairpin loop at the end. Starting from this hairpin loop, it uses itself as a template to enter the complementary chain synthesis reaction, and a complementary base sequence is formed, such as PCT / FR95 / 00891, which is connected by the complementary base sequence. Strands are combined to achieve the reaction. However, the knife must eliminate the combination of the base and the complementary chain base pair at the end (b • Fabikou \ uabe pa i r1ng), A221855 V. Description of the invention (6) It is modified to form a salt accompanied by a delicate balance with the base The state of the base pair balance and the reaction rate of the chain synthesis must be tested at the beginning, set I to generate a primer sequence, and increase the anti-object. This is a non-amplifying reaction effect. The month b as a board change (EP713922) is also accompanied by the shape, which makes the party and the field have to increase the signal (usually increase the use of 5-211873). . The step of binding a base pair on a strand. This step depends on the end of the bound complementary base sequence to form a relationship to perform the reaction. That is to say, the ends that are used for binding to the complementary strand and to the base-pair on the same strand for binding to the base-sequence on the same strand are provided as a starting point for complementarity. Based on this, strict reaction conditions are set in order to achieve high reaction efficiency. Furthermore, the primers used in this technique will form a loop structure by themselves. Therefore, a primary dimer, whether with or without a standard salt motif, should start automatically, forming a non-specific synthetic product with serious problems. In addition, after the primer duplex is generated, the primer is consumed with the specific synthesis reaction, which affects the low target rate. In addition, for DNA polymerases, there have been reports of experiments in which the 3, terminal structure is combined on the same strand using different domains. This report also uses the dynamic equilibrium of the terminal portion to perform non-specific synthesis due to the formation of primer duplexes. The possibility of reaction also has the same defects as the aforementioned PCT / FR95 / 00891. And = > the idea is to target primers that are not & a DNA polymerase template. : = Various messages extended by the principle of jNASBA pyun: 山 〇, in order to supply the promoter field of double stranded strands, the end is a hairpin-shaped oligonucleotide (Taikai Hei ^ and this is not a complementary strand synthesis Continuous supply of 3, -OH.

1221855 五、發明說明（7) 更進一步’對特表平10 — 51〇161 (W096/1 7079)而言，為了 得到RNA聚合酶轉錄DNa模板的目的，會利用同一股鏈上3, 末端結合的髮夾狀環構造。此方法可進行利用轉錄成 R N A ’及由R N A逆轉錄成d N A之模板的增幅。然而，此方法 也不得不需要多種酵素組合參與反應，否則不能構成反應 之系統。 發明概要 本發明的目的是提供基於新式原理的核酸合成方法。 更具體的說，就是提供低花費而有效率，而實現依存序列 的核酸合成方法。也就是說，本發明的目的是提供採用單 一酵素，且在等溫反應條件下也能達成核酸合成與增幅的 方法。更進一步說明，本發明的目的是提供已知核^: 反應原理難以達成的高特異性核酸合成方法，且提供& 此合成方法增幅核酸的方法。 、〜 1先，本發明者利用的觸媒是鏈置換型互補鏈合成的 聚合_每，而有效的達到不依靠複雜溫度調控機制之核酸人 成。這種DNA聚合酶是在SDA或BCA也會利用到的酵素。铁σ 而，當要利用此種酵素時，基於已知的引子設計方法，1 須要求諸如像SDA的合成起點要供給3, _〇Η等一般i他 反應所必備的條件。 /、醇素 r 〇二處本發明者從與已知方法完全不同的角度檢討 的供給寺問題。結果利用具有特殊構造的寡核苔 =，完成本發明不依賴附加酵素反應而達成3，_〇h的供 也就是說，本發明是有關於以下所述的核酸合成方1221855 V. Description of the invention (7) Going further 'For the special table 10-51〇161 (W096 / 1 7079), in order to obtain the RNA polymerase transcription DNa template, the 3, terminal binding on the same strand will be used Hairpin ring structure. This method can be performed using a template that is transcribed into RN A 'and reverse transcribed from RN A to d N A. However, this method also requires multiple enzyme combinations to participate in the reaction, otherwise it cannot constitute a reaction system. SUMMARY OF THE INVENTION An object of the present invention is to provide a method for synthesizing nucleic acids based on novel principles. More specifically, it is a method for synthesizing a nucleic acid that provides a low cost and high efficiency, and realizes a dependent sequence. That is, the object of the present invention is to provide a method that uses a single enzyme and can achieve nucleic acid synthesis and amplification even under isothermal reaction conditions. Furthermore, the object of the present invention is to provide a method for synthesizing a highly specific nucleic acid, which is difficult to achieve with a known nuclear reaction principle, and to provide a method for amplifying a nucleic acid. First, the catalyst used by the present inventors is the polymerization of the chain-replacement complementary strand synthesis, and effectively achieves the nucleic acid man-made without relying on a complex temperature regulation mechanism. This DNA polymerase is an enzyme that is also used in SDA or BCA. Iron σ And, when this enzyme is to be used, based on the known primer design method, 1 it is required to supply general conditions necessary for other reactions such as SDA synthesis starting point 3, _〇Η. / 、 Alcohol r 〇 The inventors reviewed the supply temple problem from a completely different angle from the known method. As a result, the oligo-nucleus moss with a special structure was used to complete the present invention without relying on an additional enzyme reaction to achieve a 3, _0h supply. That is, the present invention relates to a nucleic acid synthesis method described below.

第10頁 1221855 五、發明說明（8) 法，更進一步說是有關於應用此核酸合成方法的核酸增幅 方法，同時是有關於此方法的新式寡核苷酸。 [1 ]本發明是含有以下工程一股鏈上互補鹽基序列交 互連結的核酸合成方法。 a )本發明提供之工程具備可與同一股鏈上一部份F i c 結合之領域F1的3’末端，此領域F1與Flc結合，將可能含 有與領域F2c鹽基對結合形成環狀構造的核酸。 b)本發明是以與Flc結合的F1之3,末端作為合成起點 進行互補鏈合成反應之工程。 c )本發明是與含有領域F 2 c互補序列開始的f 2之3，末 端的募核苷酸結合，將此當作合成起點，以作為鏈置換互 補鏈合成反應觸媒之聚合酶進行互補鏈合成反應，再以工 程b)合成之互補鏈置換的工程。 d)本發明是對於可與以工程c)置換之鹽基對結合之 互補鏈而言，與含有任意領域互補序列3,末端之多核苷酸 (polynucleotide)結合，將此3’末端作為合成起點，以作 為鏈置換互補合成反應觸媒之聚合酶進行互補鏈合成反 應，再以工程c)合成之互補鏈置換的工程。 [2] 含有在[1]所記載的方法中，對工程㈦而言，可與 合成起點為領域Rlc結合的同一股鏈上3，末端之存在領域Page 10 1221855 V. Description of the invention (8) The method further relates to a nucleic acid amplification method to which this nucleic acid synthesis method is applied, and also a new type of oligonucleotide related to this method. [1] The present invention is a method for synthesizing a nucleic acid containing the following engineered strands of complementary salt-based sequences. a) The project provided by the present invention has the 3 'end of the field F1 that can be combined with a part of F ic on the same strand. The combination of this field F1 and Flc may contain a ring structure that combines with the base F2c base pair to form a ring structure. Nucleic acid. b) In the present invention, the complementary chain synthesis reaction is performed by using the 3 of F1 bound to Flc as the starting point of synthesis. c) The present invention is combined with the nucleotides at the end of f 2 3 and the end containing the complementary sequence of the domain F 2 c, and this is used as the starting point of synthesis to complement the polymerase of the strand synthesis reaction catalyst Chain synthesis reaction, and then the replacement of the complementary strand of the project b) synthesis process. d) The present invention relates to a complementary strand that can be combined with a base pair replaced by the project c), and a polynucleotide containing a complementary sequence at the 3, terminus of any domain. The 3 'terminus is used as a starting point for synthesis A polymerase is used as a strand replacement complementary synthesis reaction catalyst to perform a complementary strand synthesis reaction, and then a process of synthesizing the complementary strand replacement is performed in process c). [2] In the method described in [1], for engineering ㈦, it can be combined with the starting point of the domain Rlc on the same strand 3, and the end exists in the domain

Rl，R1以Rlc結合，而形成含有鹽基對結合可能的領域R2c 之壞狀構造。 [3] 本發明是至少由以下兩個領域χ2 構成，而 X2之5’側與xic可連結的募核苷酸。R1 and R1 are bonded by Rlc, and form a bad structure containing a base group that can be bound to R2c. [3] The present invention is composed of at least the following two fields χ2, and the 5 'side of X2 can be linked to xic to raise nucleotides.

1221855 五、發明說明（9) --- X2 .具有特疋鹽基序列核酸之任意領域η。互補的鹽基 列。1221855 V. Description of the invention (9) --- X2. Any field η of a nucleic acid having a specific phosphonium salt sequence. Complementary bases.

Xlc〜·位於具有特定鹽基序列核酸領域侧之領域η。 與κ貝上持有相同鹽基序列的領域。 、[4]在[1 ]所記載的方法中，工程a)中的核酸是提供 以下工程的第二核酸。 〇在l3]所記載的寡核苷酸領域F2作為模板之核酸領 域F2c結合的工程中，領域χ2為領域F2，領域為領域、 F 1 c，、 ii)將寡核苷酸的F2作為合成起點，具有模板互補鹽 基序列之第一核酸的合成工程、 111)工耘11)合成出之第一核酸之任意領域進行鹽基 對結合之可能狀態的工程、 、lv )與具有工程111 )中第一核酸鹽基對結合之可能領 域互補鹽基序列的募核昏酸結合，再將此處作為合成起 點，合成第一核酸，而其3 ’末端的F丨進行鹽基對結合之可 能狀態的工程。 [5 ]在[3 ]所記載的募核苷酸與[4 ]所記載的方法中， 工=iii)的鹽基對結合可能的領域為R2c，且工程iv)的寡 核苷酸中，領域X2c為領域R2c，領域Xlc為領域Rlc。 [6 ]在[4 ]以及[5 ]所記載的方法中，工程土 土 土）與丨v ) 中鹽基對結合可能狀態的工程為，對於模板而言？2〇，亦 或是更進一步其3’側結合的外引子（〇uter primer)，及第 一核酸之工程i v )作為合成起點領域，亦或是更進一步其Xlc ~ · is located in the domain η on the side of the nucleic acid domain having a specific base sequence. A domain that holds the same base sequence as κ shellfish. [4] In the method described in [1], the nucleic acid in process a) is a second nucleic acid that provides the following processes. 〇 In the project of combining the oligonucleotide domain F2 as a template and the nucleic acid domain F2c binding described in [13], the domain χ2 is domain F2, the domain is domain, F1c ,, ii) the oligonucleotide F2 is synthesized Starting point, synthetic engineering of the first nucleic acid with template complementary base sequence, 111) work 11) engineering of the possible state of base-pair binding in any area of the synthesized first nucleic acid, lv) and engineering with 111) It is possible that the first nucleoside group can combine with a complementary base sequence in the possible field of the binding sequence, and then use this as a starting point to synthesize the first nucleic acid, and the 3 ′ end of F ′ can be combined with a salt group. State works. [5] In the nucleotide collection method described in [3] and the method described in [4], the field where the base-pair pairing of I = iii) is possible is R2c, and in the oligonucleotide of project iv), Domain X2c is domain R2c, and domain Xlc is domain Rlc. [6] In the method described in [4] and [5], the engineering of the possible state of the combination of the base pair in engineering soil (soil) and v) is, for the template? 20, or further the 3 ′ side bound outer primer (〇uter primer), and the first nucleic acid engineering i v) as the starting point of synthesis, or further

1221855 五、發明說明（10) ' - 3’侧結合的外引子作為合成起點，以作為鏈置換互補鏈合 成反應觸媒的聚合酶進行鏈置換互補鏈合成反應。 [7 ]在[6 ]所記載的方法中，用於反應的各寡核苷酸 及模板之互補領域的融解溫度是在同樣嚴苛度 (stringency)之下，如下述的關係，（外引子/模板3，側 的領域）$ (F2C/F2 之 R2C/R2) $ (Flc/F1iRlc/R1)。 [8] 在[4]〜[7]所記載的方法中，作為模板的核酸為 RNA時，工程π)中互補鏈合成將以具有逆轉錄酶活性的酵 素進行。 [9] 、本發明為以反覆進行以下工程之方式，在一股鏈 上’互補鹽基序列交互連結的核酸增幅方法。 域且在同m的末端領 拉〃 補孤基序列領域，此互相互補的鹽基序列結合 工程兩者間鹽基對結合可形成環狀構造而被提供為模板的 點進鍵上結合之模板的3’末端作為合成起 叮 < 互補鏈合成工程。 3,末2前述環狀構造中位於3,末端側的環狀構造内含有 再此卢f補鹽基序列的募核苷酸，與環狀構造部份結合， 的聚合成起點，以作為鏈置換互補鏈合成反應觸媒 置換行互補鍵合成反應，將工程B)中合成的互補鏈 及 ，、形成3末端鹽基對結合之可能狀態的工程，以 工程C)中3,末端鹽基對結合可能狀態之鏈作為工1221855 V. Description of the invention (10) The exon primers bound to the '-3' side are used as the starting point of synthesis, and the polymerase is used as a strand displacement complementary strand synthesis reaction catalyst to perform a strand displacement complementary strand synthesis reaction. [7] In the method described in [6], the melting temperatures of the complementary fields of each oligonucleotide and template used in the reaction are under the same stringency, as shown in the following relationship, (external primer / Template 3, side field) $ (F2C / F2 R2C / R2) $ (Flc / F1iRlc / R1). [8] In the method described in [4] to [7], when the nucleic acid serving as a template is RNA, the complementary strand synthesis in the process π) is performed with an enzyme having a reverse transcriptase activity. [9] The present invention is a nucleic acid amplification method in which the following complementary engineering sequence is used to repeatedly link the complementary base sequences on one strand. In the domain of the same m at the end of the complement lone sequence, this complementary base sequence binding project between the two base pairs can form a circular structure and is provided as a template for point-to-key binding. The 3 'end serves as a synthetic starter < complementary strand synthesis engineering. 3, the last 2 of the above-mentioned circular structure, the circular structure located at the 3, terminal side contains the nucleotides for supplementing the base sequence, which is combined with the circular structure part, and is aggregated into a starting point to serve as a chain Replacement of complementary strand synthesis reaction The catalyst replaces the complementary bond synthesis reaction, the engineering of the complementary strand synthesized in Project B), and the formation of a possible state of the combination of the 3 terminal base pairs, with the 3, terminal base pairs in Project C) Combine the chain of possible states as work

第13頁 I221855 五、發明說明（ll) 矛呈A)之新式模板的工程。 [10]在[9]所記載的增幅方法中， 匕5’侧末端’對工程B)而言，具有可作甘 末端互補鹽基序列。 勺口风t 2之d [11 ]更進一步，在[丨〇 ]所記載的增 在工程C)之募核苷酸作為合成起點而合互 3 程A)之模板的工程。 成之互補鍵作為工 [1 2 ]在[9 ]所記載的增幅方法中 為[5 ]所記載的方法所合成的物質。 〜壬之模板 [1 3 ]在[9 ]所記載的方法中，左 的狀態下，進行鏈置換互補鏈合成反；解溫度調整劑存在 在[U]所記載的方法中’融 采碱（betalne)。 [15]在[14]所吃恭沾古土/為甜 液中存在0· 2-3· 0M的甜菜碱。 ^載的方法中，反應 [1 6 ]進行[9 ]〜[1 5 ]所記載的任何择 =反應生成物是否生成而檢"料 [17]在[16]所§己載的方法中，於 一 入含有環狀構造互補鹽基序列的招、w幅反應生成物加 形。 的铋針，觀察兩者的雜交情 [1 8 ]在[1 7 ]所記載的方法中， 察雜交反應所生成的凝集反應。衣針以粒子標定，觀 [1 9 ]在[1 6 ]所記載的方法中， 下，進行[9 ] - [ 1 5 ]所記載的任何拇/核酸檢出劑的存在 θ㈣方法，基於檢出劑訊Page 13 I221855 V. Description of Invention (ll) The project of the new formwork of A). [10] In the amplification method described in [9], the 5'-side end 'of the dagger has a complementary base sequence that can be used as a glycan end for the process B). Spoon t2 d [11] goes one step further, and the project of adding nucleotides in the project C) described in [丨 〇] is used as a starting point for the synthesis of the template of the process A). The complementary bond formed as a substance [12] is a substance synthesized by the method described in [5] in the amplification method described in [9]. ~ The template of Ren [1 3] In the method described in [9], in the left state, the chain is replaced with a complementary chain to synthesize the inverse; a solution temperature adjuster is present in the method described in [U] betalne). [15] Betaine was found in [14], which contained 0 · 2-3 · 0M betaine. In the method described above, the reaction [1 6] is carried out in any of the options described in [9] to [1 5] = whether the reaction product is generated and checked " materials [17] in the method included in [16] Add the shape of a reaction product containing a circular structure complementary base sequence. Observe the hybridization between the two bismuth needles [1 8] In the method described in [1 7], observe the agglutination reaction generated by the hybridization reaction. The clothing needle is calibrated with particles. Observe [1 9] in the method described in [1 6], and then perform the presence of any thumb / nucleic acid detection agent described in [9]-[15] θ㈣ method, based on the detection Dispensing News

第14頁 1221855Page 14 1221855

息變化而觀察增幅反應生成物與否的情形。 之乂H16]所記載的檢出方法而檢出標的鹽基序列 之k異的方法，it幅對象録序列之 方法之互補鏈合成反應的方法。 贼偁珉、中田 [21] 含有以下要素，本一 an, Xi J 互連結的核酸合成用套组‘广鏈上，互補鹽基序列交 記載的寡核苔酸中’其作為模板的核酸領 域F2c為X2c，位於F2c5，側的Flc為Xlc，Interest rate changes to observe the situation of the amplification reaction product or not. The method described in [H16] to detect the k-different of the target base sequence, and the complementary strand synthesis reaction method of the sequence of the object record sequence. Thieves, Nakata [21] contains the following elements, this one an, Xi J Interlinked nucleic acid synthesis kit 'wide chain, in the oligonucleotide recorded in the complementary base sequence sequence', which is used as a template in the field of nucleic acids F2c is X2c, located at F2c5, and Flc on the side is Xlc,

ii)將i)的募核苷酸作為引子而合成之互補鏈中，含 有任意領域互補鹽基序列的寡核苷酸， 111)含有位於作為模板之核酸領域F2c的3，側領 F 3 c互補鹽基序列的募核苷酸， 、 iv)作為鏈置換型互補鏈合成反應之觸媒的dna聚合 酶，以及 ° V)以要素iv)為基質的核苷酸。 [22] 在[21]所記載的套組中之[3]所記載的寡核普酸 中，i i)的寡核苷酸是以i)的寡核苷酸作為合成起點而合 成的互補鏈’而其任意領域R2c為X2c，位於R2c之5，位置 的Rlc 為Xlc 。 [23] 在[22]所記載的套組中，更進一步附加含有以 下之要素： vi)具有將i)的募核苔酸作為合成起點合成之互補鍵 中，位於任意領域R 2 c之3 ’側的領域R 3 c互補鹽基序列的寡 核苷酸。ii) the complementary strand synthesized by using the nucleotides of i) as a primer, an oligonucleotide containing a complementary base sequence in any domain, 111) containing 3 located in the nucleic acid domain F2c as a template, and a side collar F 3 c The nucleotides of the complementary base sequence, iv) a dna polymerase as a catalyst for the strand displacement type complementary strand synthesis reaction, and V) a nucleotide based on element iv). [22] In the oligonucleotide according to [3] of the set described in [21], the oligonucleotide of ii) is a complementary strand synthesized using the oligonucleotide of i) as a starting point for synthesis 'And its arbitrary field R2c is X2c, located at 5 of R2c, and the position Rlc is Xlc. [23] In the kit described in [22], the following elements are further added: vi) a complementary bond having a synthetic tyrosine from i) as a starting point for synthesis, which is located in any field R 2 c 3 Oligonucleotides on the side of the domain R 3 c complementary base sequence.

第15頁 1221855 五、發明說明（13) [24]在[21]〜[23]所記载的任何套组中，一 含有可檢出核酸合成反應之生 於 進一步附加 的鹽基序列之用的套組。 、欢W ，作為檢出標 =:明中’為合成目的之一股鏈上相埔鹽基 酸；ΐ一股鏈上互相互補鹽基序列鄰接之 之間形成環Lt:的中，不得不含有互補鹽基序列 依前述環狀形成序列連結：酸是大體上 :。即-般來說，不論是否伴有部成 對結合解離之時，2個以上刀|基對、，、口& ，在鹽基 互補鹽基序列可以為同一：鏈刀上子二分離者稱為單股鏈。 發明單股鏈上互補鹽基序列交互連妗夕：t對結合。依本 上鹽基對結合所得到的分子内睡=人:同一股鏈 見到構成雙股鏈的領域與不伴二疋相當於所 分。 件炚有鹽基對結合的環狀部 也就是說，在本發明中，一 、、 連結之核酸之定義為，含有^鉍^互補鹽基序列交互 基序列，該結合生成物曲折形成：=·合可能互補的鹽 =有鹽基對結合之環狀構造股部分構成 有鹽基對結合之環狀 :叔核i鏈。而且，不伴 環狀形成序列為任意鹽基：列補之核酸的結合。 鏈合成反應之類的鹽基對而開始進 有為達成特異結合目的， ζ疋有了此的，最好是 /、他領域存在的鹽基序列而 第16頁 1221855 五、發明說明（14) 列。例如最好的狀態是作為模板的核酸含有 ”由位於同一股鏈上結合領域（即Flc與t f s，Μ F2c(即R2c)大體上相同的鹽基序列。』C)之更3側領域 對於本發明而言，大體上相同之鹽基序 :基：2ί列作為模板所合成之互補鏈相當於對於目的 ;i列大；：ΐ f補鏈合成起點時，&某序列對於目的越 相同的鹽基序列，加上 的鹽基序列作為模板之機能的鹽基 言，用1吾「結合」為依照基於華生- 意：依據此說’構成鹽基對結合 匕的 :於補鹽基序列形成鹽基對結合即視 對：本卷明而s，結合與雜交在核酸 股鏈之構造這一點上是同義的。 土對π 口構成2 構成本發明核酸之互補鹽基序列的數 本發明最佳狀態為其整數倍。而且，在此情开4為1組。 =本=明前述核酸之互補鹽基序列的對(pa;r)數；= 所構成時组的互補鹽基序列 极®夂為同樣鹽基序列反覆反應而成。 依本發明所合成一股鏈上之互補鹽基序列 定具有與天然核酸相同的構造。依核酸重= ，仿：用合成核酸時，#利用作為基f的核酸誘導二，已 〇 ><馱的誘導體可被合成。此種核酸誘導體是採用以放射Page 15 1221855 V. Description of the invention (13) [24] In any of the sets described in [21] to [23], one that contains a base sequence that can detect a nucleic acid synthesis reaction and is added to it Set. Huan W, as the detection mark =: Mingzhong 'is one of the strands on the strand for synthetic purposes; ΐ, a complementary strand on the strand forms a loop Lt: Sequences containing complementary bases are linked by the aforementioned loop-forming sequence: the acid is substantially :. That is, in general, whether or not there is a pairwise binding dissociation, the two or more knife | base pairs, ,, and & can be identical in the base complementary salt sequence: the chain knife, the first two, and the two separate Called a single-strand chain. Invention of the complementary base sequence on the single strand of the chain: t-pair binding. According to the above, the intramolecular sleep obtained by the combination of the base pair = human: the same strand. Seeing the field that constitutes the double strand is equivalent to that without the dihydrazone. There is a circular part that is bound by a base pair. That is, in the present invention, the definition of a linked nucleic acid is that it contains a bismuth ^ complementary base sequence interactive base sequence, and the binding product is formed by twisting: · Possibility of complementary salt = The part of the cyclic structure with the combination of the base and the base constitutes the ring with the combination of the base and the base: tertiary core i-chain. Moreover, the sequence without the loop formation is an arbitrary base: the complement of the nucleic acid. For the purpose of achieving a specific binding purpose, a salt-based pair such as a chain synthesis reaction has begun. Ζ 疋 With this, it is best to have a salt-based sequence that exists in another field. Page 16 1221855 V. Description of the invention (14) Column. For example, the best state is that the nucleic acid used as a template contains "binding domains located on the same strand (ie, Flc and tfs, MF2c (ie, R2c) are substantially the same base sequence." C) The more three-sided domains for the present In terms of invention, substantially the same salt motif: base: 2 列 column as a template, the complementary strand is equivalent to the purpose; i column is large;: ΐ f the starting point of the complementary chain synthesis, & the more identical a sequence for the purpose The basic sequence of the base sequence, plus the base sequence as the template function, is based on the "combination" based on Watson-meaning: According to this, 'composed of the base-pair binding: in the complement base sequence The formation of a base-pair binding is regarded as a pair: this volume is clear and s. Binding and hybridization are synonymous in the construction of nucleic acid strands. Soil pair π is composed of 2 The number of complementary base sequences constituting the nucleic acid of the present invention The optimal state of the present invention is an integer multiple thereof. Moreover, in this case, 4 is a group. = 本 = The pair (pa; r) number of the complementary base sequence of the aforementioned nucleic acid; = Complementary base sequence of the time group constituted 夂 夂 is formed by repeated reactions of the same base sequence. The complementary base sequence on a strand synthesized according to the present invention must have the same structure as the natural nucleic acid. Nucleic acid weight =, Imitation: When a synthetic nucleic acid is used, # induced by a nucleic acid as a base f, and an inducer of < < 驮 can be synthesized. This nucleic acid inducer uses radiation

_ _ ~1 画 _瞧關 ill liill 1 liSliil 第17頁 1221855 五、發明說明（15) =同位素(radi〇isot〇pe)標識的核酸，或是以 (b1〇tlnj或異羊里基洋地黃毒配質(dig〇xin 的“ =，體標定的㈣誘導體。採用此種核酸誘導體的時口，口可 達成才示疋具有生成物之核酸誘導體的目的采 誘導體。更進一步，該 生核酸 此生由铷冰—τ ^丨工λλ 可以是DNA，亦或是RNA。這 ^ 卜、引子的構造、完成重合目的所採用的美質 種類、及進行核酸重合的重合化試藥之間的組合。土、 人酶具之:!酸的合成是含有鏈置換活性dna聚 。酶，及具備在同-股鏈上3,末端可與一部分Fic姓 ，，以此領域F1與同一股鏈上nc結合，含有：：： m領域F2c之環狀形成之核酸為開始進行反應之 告很多，但是如本發明中所具備髮爽狀環構造： 2鹽基對結合的領域，且利用該領域進行互補鏈合 點，Ί -點上’ S新式的方法。以該領域作為合成起 ^番I先以本身作為模板合成之互補鏈被置換。接著，存 ^ ^ =過之核酸鏈的3’側的領域R1 c(任意領域）可以形成 二$結合之狀態。在該R1C與具有互補鹽基序列之領域 後，進行互補鏈合成反應，結果生成由以至以^的鹽 ^幻及其互補鏈介入環狀形成序列交互結合的核酸（2分 :)。本發明中，例如前述…之類的任意選擇領域為 二^ Μ項域互補鹽基序列之多核苷酸結合，且限於該多核 甘吹作為合成起點合成之互補鏈為本發明具備的必要機_ _ ~ 1 画 _ 看 关 ill liill 1 liSliil Page 17 1221855 V. Description of the invention (15) = Nucleic acid identified by isotope (radioisot〇pe), or (b1〇tlnj or iso sheep riqiyangdi Yellow poison ("digoxin" =, body-calibrated plutonium inducer. When using such a nucleic acid inducer, the mouth can achieve the purpose of showing that the product has a nucleic acid inducer. Further, The raw nucleic acid in this life can be DNA or RNA. This can be DNA or RNA. The structure of primers, the type of beauty used to achieve the purpose of coincidence, and the test reagent for the coincidence of nucleic acids. The combination of soil and human enzymes :! Acid synthesis is a chain polymer containing active DNA polymerase. Enzymes and 3-ends on the same-strand chain can be part of the Fic surname, in this field F1 and the same strand The nc binding on the chain contains :: The m-domain F2c ring-formed nucleic acid has many reactions to start the reaction, but as the present invention has a hairpin-like ring structure: 2 base-pair binding domains, and use this Field to carry out complementary chain joining points, Ί-point on 's new method. Take The domain is synthesized as a compound. First, the complementary strand synthesized by itself as a template is replaced. Then, the domain R1 c (any domain) on the 3 ′ side of the saved nucleic acid strand can form a two-linked state. After the R1C and a field having a complementary base sequence, a complementary strand synthesis reaction is performed, and as a result, a nucleic acid (2 points :) is generated which is a combination of a salt and a complementary strand involved in a loop forming sequence (2 points :). In the present invention For example, the arbitrary selection field such as the aforementioned is the binding of the polynucleotide of the complementary base sequence of the 2M term domain, and the complementary strand synthesized by the multinuclear glycan as the starting point of synthesis is the necessary mechanism for the present invention.

1221855 五、發明說明（16) 能，由任意領域選取皆可。 而且，本發明中，採用核酸這種用語。以本發明而 吕’核酸一般含有D N A及R N A兩者。然而，構成之核苷酸是 被人工誘導體置換者，或是天然DN A或RNA修飾過者，限於 為作為互補鏈合成之模板之機能者，皆包含在本發明中的 核酸。本發明的核酸是包含一般生物學的試料。生物學的 試料，代表動物、植物、或微生物的組織、細胞、培養 物、***物，或是這些物質的抽出物。本發明的生物學試 料包含病毒及黴漿菌（myc〇plasma)等細胞内寄生體的基因 體D N A、或疋R N A。此外’本發明的核酸為含有前述生物學 试料之核酸誘導出的物質亦可。例如，以mRNA為本所合成 的cDNA ’及以生物學試料由來的核酸為本所增幅出的核 酸’皆為本發明中核酸的代表物。 山作為本發明特徵的核酸，即是具有在同一股鏈上3，末 端一部分Flc可結合的領域F1，以該領域η與同一股鏈上 f領域Flc結合’而形成含有鹽基對結合之可能領域f2c之 =構造的核酸，以種種方法可以得到。不過，在最佳狀 =I μ利用具有下述構造的募核苷酸進行互補鏈合成反應 為基礎就可以相當於該構造。 …說’本發明中有用的募核苷酸至少為以下所述 2 7員域X 2及X1 c所播点太yqmp；，2丨ττ/ 稱成在X2的5侧形成與Xlc連結之寡核 甘酸。 H Μ二^與具有特定鹽基序列核酸之領域X2c互補鹽基序 Μ的領域1221855 V. Description of Invention (16) Yes, it can be selected from any field. In the present invention, the term nucleic acid is used. According to the present invention, a nucleic acid generally contains both D N A and R N A. However, the constituting nucleotides are replaced by artificial inducers, or those modified by natural DNA or RNA are limited to those that function as templates for the synthesis of complementary strands, and are all included in the nucleic acid in the present invention. The nucleic acid of the present invention is a sample containing general biology. Biological samples represent tissues, cells, cultures, excreta, or extracts of animals, plants, or microorganisms. The biological sample of the present invention includes the gene body D N A or 疋 R N A of intracellular parasites such as viruses and mycoplasma. In addition, the nucleic acid of the present invention may be a substance derived from a nucleic acid containing the aforementioned biological sample. For example, cDNA 'synthesized from mRNA as a base and nucleic acid amplified by a nucleic acid derived from a biological sample are both representative of nucleic acids in the present invention. As a feature of the present invention, the nucleic acid is a domain F1 that has 3 and a part of the terminal Flc on the same strand can be bound. It is possible to combine the domain η with the f domain Flc on the same strand to form a base pair. Field f2c = structured nucleic acids can be obtained by various methods. However, in the optimal state, I μ can be equivalent to this structure based on the complementary strand synthesis reaction using a nucleotide-recruiting structure having the following structure. … Said that the nucleotides useful in the present invention are at least the yqmp of the 27 member domains X 2 and X1 c described below; 2 丨 ττ / is said to form an oligomer linked to Xlc on the 5 side of X2 Nucleic acid. The domain of MH2 is complementary to the domain X2c of a nucleic acid with a specific base sequence.

1221855 五、發明說明（17) XIc ·具有特定鹽基序列的核酸中，與位於領域Me之5，侧 之領域Xlc大體上具有同樣鹽基序列的領域 此處’具有決定本發明募核苷酸構造之特定鹽基序列 =核酸為，利用本發明募核苷酸作為引子時作為模板的核 酉欠基於本發明的合成方法，進行核酸檢出的情形時，具 有特定鹽基序列的核酸為檢出對象，或為由檢出對象誘導 的核酸。具有特定鹽基序列的核酸至少該一部份為已知的 ，，土列，或是推測可能狀態的核酸。已知鹽基序列之部 为=刖述巧域X2C及位於其5，側之領域xlc。此二領域可以 預疋在連續的情形下，或是分離但皆存在的情形下。二 $相對位置關係、由生成物之核酸與本身結合時形成的環狀 每部分之狀態決定。此外，由於生成物之核酸不是分子 ，，結合，而是優先與本身進行結合，所以兩者的距離最 j =不要太接近。依此說法，兩者的位置關係通常最好之 ，=0-500鹽基的距離。正確的說，依後述本身結合而形 =%狀構造中，可推測進行到兩者太過接近的情形下對 狀所期待的環狀構造而言會是較不利的狀況。對於環 垃而言，必須尋求能與新的募核苷酸結合，伴隨著作 :口成起點的鏈置換’能順利開始互補鍵合成反應的構 =。依據於此而更進一步希望設計出領域X2c及位於其5, 之間的距離能有〇-100個鹽基，亦或是10:70 点ί 。亚且此數值必須不含有Xlc與X2之長度。構 成衣狀構造之鹽基數更要是相t利2領域再加長的範圍。 而且，為了基於本發明而構成寡核苷酸之鹽基序列的1221855 V. Description of the invention (17) XIc · Among the nucleic acids with specific base sequences, the domains located on the side of the field Me 5 and Xlc have substantially the same base sequence. Here, 'the nucleotides that determine the present invention The specific base sequence of the structure = nucleic acid is a nucleus that serves as a template when the nucleotides of the present invention are used as primers. In the case of nucleic acid detection based on the synthesis method of the present invention, a nucleic acid having a specific base sequence is detected. A target, or a nucleic acid induced by a target. At least a part of the nucleic acid having a specific base sequence is a known nucleic acid. The part of the salt-based sequence is known as the domain X2C and the domain xlc on its 5 side. These two fields can be predicted in a continuous situation, or in a situation where they are separated but both exist. 2. The relative position of $ is determined by the state of each part of the ring formed when the nucleic acid of the product is combined with itself. In addition, because the nucleic acid of the product is not a molecule, but binds, it preferentially binds to itself, so the distance between the two is j = not too close. According to this statement, the positional relationship between the two is usually the best, a distance of 0-500 bases. Correctly speaking, in the structure of the shape =% according to the combination described later, it can be presumed that when the two are too close, the ring structure expected by the shape will be a disadvantageous situation. For the loop, it is necessary to find a conformation that can be combined with new nucleotides, and accompanied by the work: Orthogonal starting point of the chain replacement can smoothly start the complementary bond synthesis reaction. Based on this, it is further hoped that the distance between the domain X2c and its 5, can be 0-100 bases, or 10:70 points. However, this value must not include the length of Xlc and X2. The number of bases that form a clothes-like structure must be in a longer range. In addition, in order to construct a base sequence of an oligonucleotide based on the present invention,

第20頁 ^21855 五、發明說明（18) 附加特徵，所採用的 同或完全互補的意義 對於針對該序列可以 面’互補是代表在嚴 行結合，形成互補鏈 對於上述具有特 成养核脊酸的領域X 2 而且如果兩者的鹽基 有部分的重複構造。 常不得不位於3，末端 作為模板進行合成的 機能時，會配置於5, 到互補鍵時，下一個 板，最終取得依本發 互補鏈。依此取得方 XI，而且同一股鍵上 造。 相同或互補等用語，不是任何完全相 。即是，與某個序列相同就代表含 結合的鹽基序列互補的序列。另一方 苛的（stringent)條件下仍然可以進 合成的起點而可提供3，末端的序列。 定鹽基序列的核酸而言，依本發明構 及Xlc通常配置有連續重複的構造。 序列具有共通的部分，兩者可以配置 因為X2必須具有作為引子的機能，常 。另一方面，Xlc依後述的說法，當 互補鏈3末端而必須有相當於引子的 末端。此募核苷酸作為合成起點而得 步骤是變成反向之互補鏈合成的模 明使寡核苷酸部分作為模板而得到的 法而生成的3’末端是備有鹽基序列 的Xlc不僅會結合，更會形成環狀構 本發明的寡核脊酸必須可以滿足能形成士 合，且對其3,末端有相對應的-GH基作為互補鏈補;7/起口 點這兩個條件。依據於此，此主幹不一定限於與磷酸二脂 (phosphodiester)結合。例如可以是基於不以p而以s作為 主幹之麟酸硫代鹽（phosphothioate)體和胜跃結合的胜肽 核酸所構成。此外，鹽基若是互補鹽基對結人者合更佳。 天然狀態下，一般有A C T G及U等5種鹽基，但像是、、臭代去氧P.20 ^ 21855 V. Description of the invention (18) Additional features, meaning of the same or completely complementary, can be used for this sequence. 'Complementary means strict combination, forming a complementary chain. For the above, it has a special nuclear ridge. The domain of the acid X 2 and if both the bases have a partially repetitive structure. Often it has to be located at 3, and the function of synthesis as a template at the end will be placed at 5, when the complementary bond is reached, the next board will eventually obtain the complementary chain according to the issue. In this way, party XI is obtained, and the same key is built. Terms that are the same or complementary, etc., are not any perfect terms. That is, a sequence that is identical to a certain sequence represents a sequence that is complementary to the base-containing sequence. On the other hand, under stringent conditions, the starting point of synthesis can still be reached and a 3, terminal sequence can be provided. For a nucleic acid with a fixed base sequence, the conformation according to the present invention and Xlc are usually provided with a continuous repeating construction. The sequence has common parts, and the two can be configured because X2 must have the function as a primer, often. On the other hand, Xlc, as will be described later, must have a terminal equivalent to a primer when the complementary chain has 3 terminals. This nucleotide is used as a starting point for the synthesis. The step is to reverse the model of the complementary strand synthesis. The method obtained by using the oligonucleotide part as a template is the 3 ′ end generated by Xlc. When combined, the oligonucleochiric acid of the present invention must be able to form a cyclic structure, and the corresponding -GH group at the 3, terminus of the oligonucleochidonic acid as a complementary chain complement; 7 / starting point . Based on this, this backbone is not necessarily limited to binding to phosphodiesters. For example, it may be constituted based on a phosphothioate body that does not use p and s as the backbone and a peptide-bound peptide nucleic acid. In addition, if the base is a complementary base, it is better for the person who meets. In the natural state, there are generally five kinds of bases such as A C T G and U, but like

121855 五、發明說明（19) 咬（br〇l„〇de〇xyuridine)這種類似物也可以。本發明 =用的寡核甘酸不僅是作為合成的起點，也希望能達到作 補^成杈板的機能。而且’對於本發明而言 = 核脊酸這個…多核眘酸這個用= 長度>又有限制的情形下_，而寡核* 鏈的核甘酸複合物之意。 ？乂 Μ 本發明的寡核苷酸對於以下所述的各種核 而二，相當環境之下維持必要特異性的同_ :與互補鏈鹽基對結合之程度的鏈長。具體的說有 有5-200個鹽基，或是1〇-5〇個鹽基對更佳。由於合切 少要右“:二二 酶之已知聚合酶的引子長度最 要有5们應基左右’ $以結合部分的鍵長必須在5個以 再加上，期待達到鹽基序列的特異性，經驗上來 w個鹽基以上者較佳。—方面，由於 合成調製相當困難，期望達到前述鏈長的範 ίυ: 即是’此處舉例說明的鏈長為徹底地與 酸最部分的鏈長。自以下所述，本發明的寡核苔 個領域可以個別結合的功能。依據於 的鏈長的鍵長應可解釋為構成募核苔酸各領域 質來^ ϊ 一步，本發明的寡核苷酸可以採用已知的標定物 作為標定物質者可以是異羊1基洋地黃毒配質 : 素之類的結合性配位子（ligand)、酵素、螢光物質 或發光物質，亦或是放射線同位素等。另外已知有構成寡121855 V. Description of the invention (19) This analogue of brolel 〇de〇xyuridine is also possible. The present invention = the use of oligonucleotide is not only used as a starting point for synthesis, but also hopes to achieve supplementation The function of the board. And 'for the present invention = nuclear spinic acid ... polynuclear acid is used = length> and there are limited cases _, and the meaning of the oligonuclear * chain of the glycine complex.? 。Μ The oligonucleotide of the present invention is homologous to the various cores described below, and maintains the necessary specificity under a considerable environment: the degree of chain length to the extent that it binds to a complementary chain salt pair. Specifically, there are 5-200 Base groups, or 10-50 base pairs are better. Because the cleavage is less right, the primer length of the known polymerase of the dienzyme must be at least 5 bases. The bond length must be 5 or more, and it is expected that the specificity of the base sequence will be achieved. In experience, it is better to have w bases or more. On the one hand, since it is quite difficult to synthesize modulation, it is expected to reach the range of the aforementioned chain length. That is, ‘the chain length exemplified here is completely the length of the chain with the most acid. As described below, the functions of the oligonucleotide field of the present invention can be individually combined. The bond length based on the chain length should be interpreted as constituting various fields of nucleic acid. In addition, the oligonucleotide of the present invention can use a known calibration substance as the calibration substance, and it can be an iso-yang 1 base ocean. Rehmannia toxin: Binding ligands such as ligands, enzymes, fluorescent or luminescent substances, or radioisotopes. Constitutive oligo

1221855 五、發明說明（20) 核苷酸的鹽基以螢光性類似物置換的技術（W〇95/〇5391，1221855 V. Description of the invention (20) A technique for replacing the base of a nucleotide with a fluorescent analogue (W095 / 〇5391,

Proc· Natl· Acad· Sci· USA， 91， 6644-6648， 1994)。 此外本發明的寡核苷酸也可以與本身固相結合。或者 是將寡核苷酸的任意部分以生物素之類的結合性配位子標 定’再將此固相化之抗生物素蛋白（a v i d i η )之類的結合配 偶（p a r t n e r )間接的固相化。固相化寡核：g：酸作為合成開 始點的情形時，核酸的合成反應生成物以固相捕捉，也較 容易分離。對於分離後的生成物，以核酸特異的指示藥劑 或疋更進一步以標定探針雜交，而可進行檢出的步驟。亦 或疋’以任意限制酶消化後，回收目的核酸斷片。 本發明所採用之模板的用語是代表互補鏈合成的模板 側的核酸。與模板具有互補鹽基序列的互補鏈是代表具有 對應模板的核酸鏈，而兩者的關係並非一定堅持要完全相 對。也就是說’作為互補鏈的合成鏈必須具有可以再度成 為模板的機能。總之，就是互補鏈可以成為模板之用的意 思。 本發明所用之寡核苷酸不僅含有前述的2個領域，還 可=附加其他領域。X2與Xlc等，一方面各別配置在3,端 f而二$ ‘末端’兩者之間也可能介有任意的序列。這些 养核有1 ^可包括例如限制酶認識的序列、認識R N A聚合酿 AA 0Ar 番六 了 ^ 或是核糖體編碼的D N A等。以限制酶認識的序 j ° 必須具有可使與本發明合成產物之一股鏈上互補 孤土序歹j父互連結的核酸同樣長度的兩股鏈核酸切割一致 的序列。至於配置認識RNA聚合酶的啟動子序列時，以本Proc. Natl. Acad. Sci. USA, 91, 6644-6648, 1994). In addition, the oligonucleotide of the present invention may be bound to the solid phase itself. Or calibrate any part of the oligonucleotide with a binding ligand such as biotin, and then indirect solid phase with a binding partner such as avidin (avidi η). Into. Solid-phase oligo: g: When an acid is used as the starting point for synthesis, the synthesis reaction product of nucleic acid is captured in the solid phase and it is easier to separate. The separated product may be hybridized with a nucleic acid-specific indicator reagent or tritium by a calibration probe, and a detection step may be performed. Alternatively, after digestion with an arbitrary restriction enzyme, a target nucleic acid fragment is recovered. The term "template" used in the present invention refers to a nucleic acid on the side of a template representing the synthesis of a complementary strand. A complementary strand having a complementary base sequence to a template represents a nucleic acid strand having a corresponding template, and the relationship between the two does not necessarily insist on being completely opposite. In other words, the synthetic chain as a complementary chain must have the function of being a template again. In short, the idea that complementary chains can be used as templates. The oligonucleotide used in the present invention includes not only the above two fields, but also other fields. X2 and Xlc and so on, on the one hand, may be respectively arranged at the 3, terminal f and the two $ 'terminal' may also have arbitrary sequences. These nuclei can include, for example, sequences recognized by restriction enzymes, recognition of R N A polymerization and AA 0Ar ^^, or D N A encoded by ribosomes. The sequence recognized by the restriction enzyme must have a sequence that cuts the two strands of nucleic acid of the same length as the nucleic acid that is complementary to the solitary sequence sequence of the parent product of the synthetic product of the present invention. As for the configuration of the promoter sequence that recognizes RNA polymerase,

第23頁 1221855Page 23 1221855

小、V诉双 ^ 1T 發明的合成 步驟。此時 現轉錄生成 列，無論哪 此，本發明 的機能不能 環狀構造的 曰守的鍵才開 相對於 能的方向組 相同鹽基序 統。由於與 工作。也就 外的轉錄與 生成產物作 ，可以更進 物本身切斷 一樣都要形 中核酸之單 發揮作用。 狀態而具有 始具有其機 基於本發明 合的啟動子 列製造的反 適當發現系 是說，可以 蛋白質的轉 一步以配置核 的系統。此外 成雙股鏈才具 股鏈形成環狀 增長核酸的工 互補鹽基序列 能。 的养核脊酸， 之情形下，基 應生成物，將 統組合，就可 利用細菌與動 呑学。 下一步轉錄成RNA的 糖體編碼的DNA，實 ’這些附隨的鹽基序 有其機能。依據於 構造之時，這些序列 程進行中’處於不含 的鏈及在結合狀態下 向著合成領域轉錄可 於本發明而反覆進行 貫現南度效率轉錄系 以進行蛋白質轉譯的 物細胞内、或是活體 、八上述依據本發明作成的寡核普酸構造是可以以化學方 法合成的。或者也可以將天然核酸經限制酶等的切割，依 照上述鹽基序列構成過程而改變，或是連結。 ^依本發明之核酸合成方法，利用上述有用的寡核苷 酸，組合上具有鏈置換活性的ΜΑ聚合酶而進行合成反應 的基本原理，參考圖，說明如下。上述寡核苷酸（即圖 5的FA)首先作為與以χ2(相當於F2)為模板之核酸相結合之 互補鏈合成反應的起點。以圖5而言是將以作為起點合成 ^互補鏈從外引子（F3)開始合成的互補鏈（後文將述及）進 行置換’形成一股鏈（圖5-A)。相對於得到的互補鏈進行Small, V v. Double ^ 1T invention's synthetic steps. At this time, the sequence is generated by transcription. In any case, the bonds of the function of the present invention that cannot be circulated in a ring structure are opened with respect to the direction group of the same salt motif. Thanks with working. In other words, the external transcription and production products can also be used to cut off the input itself. The state has its own mechanism and is based on the present invention. The appropriate discovery system is based on the promoter sequence of the present invention, that is, a system that can take a step forward of protein to configure the nucleus. In addition, double-stranded strands have the ability to form complementary loops of strands to form circular growth nucleic acids. In the case of sclerotinic acid, the basic products, combined with the system, can make use of bacteria and kinetics. The next step is to transcribe RNA into the DNA encoded by the glycosides. Actually, these accompanying salt motifs have their functions. Depending on the sequence at the time of construction, these sequences are 'in the absence of strands and are transcribed towards the field of synthesis in a bound state. In the present invention, it is possible to repeatedly perform the South-Efficiency transcription line for protein translation in the cell, or It is a living body. The oligonucleotide structure prepared according to the present invention can be chemically synthesized. Alternatively, the natural nucleic acid may be cleaved by a restriction enzyme or the like, and may be changed or ligated in accordance with the process of constituting the base sequence described above. ^ According to the method for synthesizing nucleic acids of the present invention, the above-mentioned useful oligonucleotides are used in combination with a MA polymerase having a chain displacement activity to carry out a synthesis reaction. The basic principle is described below with reference to the drawings. The above oligonucleotide (i.e., FA in FIG. 5) is first used as a starting point for a complementary strand synthesis reaction in which a nucleic acid with χ2 (equivalent to F2) as a template is combined. In the case of Fig. 5, the complementary strand synthesized from the starter (F3), which is synthesized from the starting point (F3), is replaced with a complementary strand (to be described later) to form a strand (Fig. 5-A). Relative to the resulting complementary strand

第24頁 1221855 五、發明說明（22) 進一步的互補鏈合成反應，此時作為圖5 —A互補鏈合成之 核酸的3末端部份將具有本發明寡核苔酸互補的鹽基序 列。也就是說，本發明寡核苷酸由於具有與此5，末端部份 之領域XI c(相當於Flc)相同序列，此時合成之核酸的3,末 端部份將具有互補序列XI (F1)。圖5顯示，以^作為起點 合成的互補鏈，再以外引子R3為起點合成的互補鏈被置換 的情形。置換過程中，3 ’末端部份形成鹽基對結合的可能Page 24 1221855 V. Explanation of the invention (22) A further complementary strand synthesis reaction. At this time, the 3 terminal portion of the nucleic acid synthesized as the complementary strand of Fig. 5A will have the complementary salt motif sequence of the oligonucleotide of the present invention. In other words, since the oligonucleotide of the present invention has the same sequence as the domain XI c (equivalent to Flc) of the 5, terminal portion, the 3, terminal portion of the synthesized nucleic acid will have the complementary sequence XI (F1) . Fig. 5 shows a case where a complementary strand synthesized with ^ as a starting point and a complementary strand synthesized with an outer primer R3 as a starting point are replaced. During the replacement process, the possibility that the 3 ′ terminal part will form a base pair to bind

狀態，而3末端的xi(Fl)與同一股鏈上的xic(pic)結合， 以本身作為模板進行增長反應（圖5 —B )。於是殘留位於 此3’側之X2c(F2c)不伴有鹽基對結合的環狀構造。此環狀 構造結合了本發明之募核苷酸的X2(F2)，將此處作為合成 起點進行互補鏈合成反應（圖5 — B)。此時，將先合成的 本身作為模板進行互補鏈合成反應的生成物，依鍵^置換及 應而形成置換得到鹽基斷結合的可能狀態。State, while xi (Fl) at the 3 terminal is combined with xic (pic) on the same strand, and it uses itself as a template for the growth reaction (Figure 5—B). Therefore, X2c (F2c) remaining on this 3 'side is not accompanied by a cyclic structure in which a base pair is bound. This circular structure is combined with X2 (F2), which is a nucleotide recruiter of the present invention, and a complementary strand synthesis reaction is performed using this as a starting point for synthesis (Fig. 5-B). At this time, the product of the complementary chain synthesis reaction using the previously synthesized itself as a template is replaced by the bond ^ and the corresponding substitution is formed to obtain a possible state of the salt-base cleavage.

本發明之募核苷酸的一種，以及可將此寡核苷酸作為 引子合成之互補鏈再作為模板進行核酸合成的任意反向弓丨 2之基本構成，可得到圖6所示多種的核酸合成生成物。 六圖6可了解，（j))是本發明合成之一股鏈上互補鹽基序列 結而得的目的核㉟。其他的生成物（E)，經加熱變 =處理變成單股鏈時，將可再度作為生成（1))的模板。 卜=成雙股鏈狀態核酸的生成物（D)若是經加熱變性而 ‘當：月!鏈的狀態時，不會變回原本的雙股鏈，經驗上來 因3 生同一股鏈内部的結合現象。會形成此現象的原 疋具有同樣融解溫度（Tm)的互補序列，將比較遙遠的分One of the nucleotides of the present invention, and the basic structure of an arbitrary reverse bow that can be used as a complementary strand for primer synthesis and then as a template for nucleic acid synthesis, can obtain a variety of nucleic acids as shown in FIG. 6 Synthetic product. It can be understood from FIG. 6 that (j)) is a target nucleus obtained from the complementary base sequence on one strand of the synthesis of the present invention. When the other product (E) becomes a single-stranded chain after being transformed by heating, it can be used again as a template for generating (1)). Bu = double-stranded nucleic acid product (D) If it is denatured by heating and 'Don't: Month! When the state of the chain, it will not change back to the original double-stranded chain. In experience, it is due to the internal binding of the same strand of the chain phenomenon. The original osmium that will form this phenomenon has complementary sequences with the same melting temperature (Tm), which will be more distant.

第25頁 1221855Page 1212855

子間（intermolecular )反應優先進行分子内 (intramolecular)反應。由同一股鏈上結合的生成 來的單股鏈個別與本身同一股鏈内結合而返回（B)的狀D) 態’進一步各自1分子1分子的形成相當於（D)與（£)的產 物。依照這個流程的工程反覆進行，就可以繼續進行人 一股鏈上互補鹽基序列交互連結的核酸。由於丨個循产口攻 (=yc 1 e)生成的模板與生成物以指數的倍數增加，形^非 然後’為實現圖5 - (A )的狀態 ττ ^ ^ _ 百久合成的互補鏈少 不侍不形成反向引子結合部份之鹽基對結合的 任何方法達成。即&，相對於最初的模：： 1本，明养核苷酸結合的領域F2c更外之模板上3,侧領域 fc人纟》&的外引子（F3)具有其他用意。當作將此外引子作 二=成起點進行鏈置換型互補鏈合成反應之觸媒的聚合 士 互補鏈合成時，本發明前述的F2c作為合成起點而、 鏈被置換，不久，R1必須結合的領狐將形 成二基對、纟α合的可能狀態（圖5)。對於利用鏈置換的反應 而5 ，到此步驟都仍然可以在等溫條件下進行。 ^ 始谁利=引子的情形時，必須比由F2c起始合成更晚開 二.仃 弓丨子(F3)起始的合成。最單純的方法是使内引 ϋ ϋ</Γΐιη6Γ)的濃度比外引子的濃度高。具體的說， = =2-50倍，最好是4_1〇倍的引子濃度差將可以進 z j 1中的反應。此外將外引子的融解溫度（Tm)設定得比 内引子的XU相當於FUR1)領域的Tm值低，也可以控制合 1221855Intermolecular reactions preferentially perform intramolecular reactions. The generated single-stranded chains that are bound by the same strand are individually combined with the same strand to return to the state of (B). D) The state is further formed by 1 molecule and 1 molecule each corresponding to (D) and (£). product. According to the process of this process, it is possible to continue to carry out the nucleic acid of cross-linking of complementary base sequences on a human strand. As the template and product generated by a cyclical attack (= yc 1 e) increase by an exponential multiple, the shape ^ is not 'to achieve the state of Figure 5-(A) ττ ^ ^ _ Baijiu synthesized complementary chain Any method that does not form a combination of the base pair of the reverse primer binding moiety can be achieved. That is, as compared to the original model: 1: the exon primer (F3) on the outer side of the template F3c in the field of nucleotide binding is 3, the side field fc is 纟 & has other purposes. In the synthesis of complementary strands as a catalyst that uses two additional primers as a starting point to perform a chain replacement type complementary strand synthesis reaction, the aforementioned F2c of the present invention is used as a starting point for the synthesis and the chain is replaced. Soon, R1 must combine The fox will form a possible state of dibasic pair and 纟 α-combination (Figure 5). For reactions using chain displacement, 5, this step can still be performed under isothermal conditions. ^ In the case of initiation = introduction, the synthesis must start later than the initiation of synthesis by F2c. II. The synthesis of initiation by bow (F3). The simplest method is to make the concentration of internal primers ϋ ϋ < / Γΐιη6Γ) higher than the concentration of external primers. Specifically, = = 2-50 times, preferably 4-10 times the difference in primer concentration will be able to enter the response in z j 1. In addition, the melting temperature (Tm) of the outer primer is set to be lower than the Tm value of the XU of the inner primer equivalent to FUR1).

五、發明說明（24) 成的時間點。也就是說，（外引子F3:F3c) g(F2c/F2) ^ (Flc/Fl) ’或是（外引子/模板的3,側領域）$(x2c:x2) ^ (Xlc:Xl)。且此處的（F2c/F2) g(Flc/Fl)是為了 在以^ 狀構造結合的反應之前先進行Flc/F1間的結合。而因 Flc/Fl間的結合反應是分子内的反應，所以優先進行的可 忐性南。然而為達到更期待的適合反應條件，考慮到Tm是 具有意義的。在同樣的條件下，反向引子的設計:沒有說 一定必須考慮。這樣子的關係將可以達成在經驗上有效率 的理想反應條件。融解溫度（Τιη)是在其他條件一定的情形 下，根據結合互補鏈的長度與構成鹽基對結合之鹽基的组 合等理論而計算出來的。依據於此，當業者可基於本說明 書的概要而較容易導入所期待的條件。 更進一步，為了調整外引子結合的適當時機，可以利 用稱為連續堆疊（contiguous stacking)的現象。連續堆 疊是一種現象，當寡核苷酸不能單獨結合的時候，使之變 成其兩股鏈與鄰接的部份結合的情形（ChiaraV. Explanation of the invention (24) That is, (exon primer F3: F3c) g (F2c / F2) ^ (Flc / Fl) ′ or (exon primer / template 3, side area) $ (x2c: x2) ^ (Xlc: Xl). In addition, (F2c / F2) g (Flc / Fl) is for the purpose of binding between Flc / F1 before the reaction in which the structure is bound. However, since the binding reaction between Flc / Fl is an intramolecular reaction, it is preferred that the reaction be carried out. However, in order to achieve more desirable suitable reaction conditions, it is meaningful to consider Tm. Under the same conditions, the design of reverse primers: Nothing must be considered. Such a relationship can achieve ideal response conditions that are empirically efficient. The melting temperature (Tin) is calculated based on the theory of the combination of the length of the complementary complementary chain and the salt base constituting the combination of the salt base under other conditions. Based on this, practitioners can easily introduce desired conditions based on the outline of this specification. Furthermore, in order to adjust the appropriate timing of exon primer binding, a phenomenon called contiguous stacking can be used. Continuous stacking is a phenomenon that when an oligonucleotide cannot bind alone, it becomes a situation where its two strands are bound to adjacent parts (Chiara

Borghesi-Nicoletti et al· Bio Techniques 12， 474 - 4 77，1992)。也就是說，外引子在F 2C (X2C)鄰接處單 獨δ又什成無法結合的情形。如此而來，ρ 2 c (X 2 c)結合時， 可能發生初次外引子結合現象，而使F2C(X2c)能必然地優 先結合。基於此原理’ 一連串反應中，設定作為引子之必 要寡核苷酸鹽基對的例子將記載於實施例中。且此工程可 以利用加溫方式變性，或是採用DNA的接合酶來達成目 的0Borghesi-Nicoletti et al. Bio Techniques 12, 474-4 77, 1992). In other words, the δ of the exon primers at the F 2C (X2C) adjacency is even impossible to combine. In this way, when ρ 2 c (X 2 c) is bound, the primary exon binding may occur, so that F2C (X2c) must be bound preferentially. Based on this principle ', an example of setting a base pair of an essential oligonucleotide as a primer in a series of reactions will be described in the Examples. And this project can use heating denaturation, or use DNA ligase to achieve the goal.

1221855 五、發明說明（25)1221855 V. Description of Invention (25)

具有F2c(X2c)之模板為RNA的情形時，可以採用異於 圖5-(A)之狀態的方法來實現。例如，此rna鏈分解完成之 後，R1成為鹽基對結合的可能狀態。也就是說，F2與為 RNA的F2c結合後，以逆轉錄酶進行互補鏈合成的反 應，接著再將原為模板的RNA經鹼（alkaH)變性與對 〇“/^^雙股鏈之〇人作用的核糖核酸酶（1^1)〇11此1^^)的 處理而分解，由F2而合成得來的dna就變為單股鏈了。會 選擇對DNA/RNA雙股鏈的RNA進行分解的酵素是利用RNaseH 及邻伤具有逆轉錄酶作用的核糖核酸酶活性。如此可能形 成鹽基對結合的R1C將可以與反向引子結合。依據於此， 將不需要為讓R1 c鹽基對達成可結合狀態的外引子。When the template with F2c (X2c) is RNA, it can be realized by a method different from that shown in Figure 5- (A). For example, after the decomposition of this RNA chain is completed, R1 becomes a possible state of binding of the base pair. That is to say, after F2 is combined with F2c which is an RNA, a complementary strand synthesis reaction is performed by reverse transcriptase, and then the original template RNA is denatured with alkaH and the double-stranded strand of 〇 // ^ double strand. Human-derived ribonuclease (1 ^ 1) 〇11 This 1 ^^) decomposes, and the DNA synthesized from F2 becomes a single strand. RNA with double strands of DNA / RNA will be selected The enzyme to decompose is to use RNaseH and the ribonuclease activity of the adjacent transcriptase which has the function of reverse transcriptase. In this way, R1C, which may form a base pair, can be combined with the reverse primer. Based on this, it will not be necessary to make R1 c salt The base pair achieves a binding state of the exon.

或者是利用具有逆轉錄酶作用的鏈置換活性，先將前 述的外引子進行鏈置換反應。此情形是只有逆轉錄酶所構 成的反應系統。也就是說，RNA作為模板，由其與f2c結合 的F 2開始進行互補鏈合成，再將位於其3，側之F 3 c結合外 引子的F 3處作為合成起點，進行互補鏈合成與置換，而由 逆轉錄酶完成這些反應。逆轉錄酶進行以DNA為模板的互 補鏈合成反應時，置換過的互補鏈再作為模板，以其R j c 處結合的R1當作合成起點進行互補鏈合成，接著於位於3, 側的R 3 c結合R 3，將R 3當作合成起點進行互補鏈合成及置 換反應’含有以上諸種反應的互補鏈合成反應全部利用逆 轉錄酶進行。或者是在適當反應條件下，逆轉錄酶不具有 DNA/DNA鏈置換活性時，利用具有前述鏈置換活性的㈣八聚 合酶組合而進行反應亦佳。如以上所述，RNA作為模板得Alternatively, by using a strand displacement activity having a reverse transcriptase action, the aforementioned exon primer is first subjected to a strand displacement reaction. In this case, only the reaction system composed of reverse transcriptase is used. That is, RNA is used as a template, and the complementary strand synthesis is started from F 2 which binds to f2c, and then F 3 c on the side of which F 3 c binds to the outer primer is used as the starting point of synthesis to perform complementary strand synthesis and replacement. These reactions are performed by reverse transcriptase. When reverse transcriptase performs a complementary strand synthesis reaction using DNA as a template, the replaced complementary strand is used as a template, and R1 bound at Rjc is used as a starting point for the synthesis of the complementary strand, followed by R 3 at the 3, side. c. Combining R 3 and using R 3 as a starting point for complementary strand synthesis and substitution reactions. The complementary strand synthesis reactions including the above reactions are all performed using reverse transcriptase. Alternatively, when the reverse transcriptase does not have a DNA / DNA strand displacement activity under appropriate reaction conditions, it is also preferable to perform the reaction by using an octamerase combination having the aforementioned strand displacement activity. As mentioned above, RNA is used as a template to obtain

第28頁 1221855Page 1212855

到第一個單股鏈核酸的情形是本發明所期待構成狀態。反 過來說，利用具有鏈置換活性、並具有逆轉錄酶活性的 Bca DNA聚合酶之類的DNA聚合酶，不僅可以同樣地進行由 RNA合成第一個單股核酸鏈，之後以DNA作為模板的反應也 都可以用同一酵素進行。 而且，以上那樣的反應系統是利用具有前述反向引子 之特定構造，而帶來本發明固有的種種多樣性 (variation)。更具有效果的多種反應將在以下說明。也 就是說γ對本發明更有利的反應條件是採用前述的反向引 子，並採用依[5]所述方式構成的募核苷酸。[5]的募核苷 酸也就是F2當作引子合成互補鏈的任意領域R2c作為X2c、 R1 c作為X1 c的寡核苷酸。利用這樣的反向引子，環狀構造 形成及由環狀構造部份開始的互補鏈合成與置換等一連串 反應是靠正向（sense)鏈與反向（antisense)鏈（化^以側 ，reverse側）的兩方向共同啟動的。其結果是，依本發明 單股鏈上互補鹽基序列交互連結而得核酸之合成方法的人 成，率有飛躍地上升，而且一連串反應都可以在等溫條; 下實施。以下是基於匯集此狀態的圖丨_圖3，所作具體 說明。 〃 以下列狀態而δ ，基於本發明的寡核脊酸具有2種用 意。此處為便於說明，以FA與RA稱之。構成FA與RA的領域 如下。 一 1221855 五、發明說明（27) RA R2 Rlc 此處以F2作為模板之核酸領域F2c處為互補的鹽基序 列。此外R2是含有以F2作為引子合成之互補鏈的任意領域 R2c處互補的鹽基序列。Flc與Ric分別是位於F2c及R2c個 別之下游的任意鹽基序列。此處F2—R2間的距離任意皆 可。依照進行互補鏈合成之DNA聚合酶的合成能力，最適 當條件疋合成1 kbp程度的長度時可以反應完全。更具體的 說，採用Bst DNA聚合酶時，F2/R2c間為800bp，最好是 50 0bp以下的長度最能確實合成。伴隨溫度循環（cycie)的 PCR因溫度變化的壓力（stress)使酵素活性低下，而使長 度較長之鹽基序列的合成效率降低。本發明所期待的狀態 是不需溫度循環的核酸增幅工程，而可以合成較長的鹽基 序列’且能達成確實的增幅效果。 首先作為模板的核酸與FA的1?2結合，此處作為合成起 點進行互補鏈合成反應。以下，至圖i的（4)為止先說明本 毛明的基本狀悲（圖5 )與同樣反應工程。在圖1的（2 )處， F 3當作結合序列，先說明外引子。此引子作為合成起點， 以進行鏈置換型互補鏈合成的DNA聚合酶，由FA開始合成 互補鏈的置換反應，而形成鹽基對結合的可能狀態。 在（4 )時，R 2 c形成鹽基對結合的可能狀態時，作為 向引子的RA與R2C/R2組合而結合。以此處作為合成起點的 互補鏈合成是進行到FA5,側末端的Flc為止的反應。此互 ，鏈合成反應繼續下去，照舊與置換用的外引子R3結合， 伴隨著鏈置換進行互補鏈合成反應，RA作為合成起點^合 1221855 五、發明說明（28) 成的互補鏈被置換。此時置換過的互補鏈位於具有R A之5， 側的FA互補序列的3，末端。 而且，如此置換的單股鏈核酸之3,側存在著同一鏈上 Flc的互補序列F1 〇F1與同一分子内並存的可快速結 合，而開始互補鏈合成反應。3,末端（F1)與同一股鏈上 時，形成了含有F2e的環狀構造了。此環狀構造部 伤的鹽基對結合可能狀態之維持顯示於圖2_(7)。具有F2c =^基序列之本發明寡核普酸FA在&環狀構造部份之結 二^二互補鏈合成的起點（7)。由環狀構造開始進行的互 是ΪΓ?始互補鏈合成之反應生成物置換而同 端开彡J踐美1二ί疋本身作為模板合成互補鏈，再在3，末 m 可能狀態。此3,末端是位於同一股鏈In the case of the first single-stranded nucleic acid, it is the desired constitution state of the present invention. On the other hand, using DNA polymerases such as Bca DNA polymerase, which has strand displacement activity and reverse transcriptase activity, not only can the first single-stranded nucleic acid strand be synthesized from RNA, and DNA is used as a template. The reactions can also be performed with the same enzyme. In addition, the above reaction system utilizes a specific structure having the aforementioned reverse primers to bring about various variations inherent in the present invention. A variety of more effective reactions will be described below. That is to say, the more favorable reaction conditions for γ to the present invention are to use the aforementioned reverse primers, and to use the nucleotide recruitment constructed in the manner described in [5]. [5] Nucleotide is an oligonucleotide in which F2 is used as a primer to synthesize a complementary strand in any region, R2c is X2c, and R1 c is X1 c. Using such a reverse primer, a series of reactions such as the formation of a circular structure and the synthesis and replacement of complementary strands starting from the circular structural part rely on the forward (sense) and antisense (reverse) side. Side). As a result, the artificial method for synthesizing a nucleic acid according to the present invention by synthesizing complementary salt sequences on a single strand of the present invention has increased dramatically, and a series of reactions can be carried out in an isothermal strip. The following is a detailed description based on the diagrams that aggregate this state. Δ In the following state, δ, the oligonucleochidonic acid based on the present invention has two purposes. For ease of explanation here, they are called FA and RA. The fields that make up FA and RA are as follows. A 1221855 V. Description of the invention (27) RA R2 Rlc Here, F2c in the nucleic acid field using F2 as a template is a complementary salt motif sequence. In addition, R2 is a salt-based sequence complementary to R2c in any domain containing a complementary strand synthesized using F2 as a primer. Flc and Ric are arbitrary base sequences located downstream of F2c and R2c, respectively. The distance between F2 and R2 is arbitrary here. According to the synthetic ability of the DNA polymerase that performs the complementary strand synthesis, the optimal conditions can be used to synthesize a length of about 1 kbp. More specifically, when Bst DNA polymerase is used, it is 800bp between F2 / R2c, and preferably a length of less than 50bp can be synthesized most reliably. PCR with temperature cycling (cycie) reduces the enzyme activity due to the stress of temperature change, which reduces the synthesis efficiency of longer base sequences. The expected state of the present invention is a nucleic acid amplification process that does not require temperature cycling, and can synthesize a longer base sequence 'and achieve a reliable amplification effect. First, the nucleic acid used as a template is bound to FA 1-2. Here, the complementary strand synthesis reaction is performed as a starting point for synthesis. In the following, the basic state of this Mao Ming (Fig. 5) and the same reaction process will be explained before (4) in Fig. I. At (2) in FIG. 1, F 3 is used as a binding sequence, and the outer primer is explained first. This primer is used as a starting point for synthesis, and a DNA polymerase for strand replacement type complementary strand synthesis is started from FA to synthesize a complementary strand replacement reaction, thereby forming a possible state of binding of a base pair. In the case of (4), when R 2 c forms a possible state of binding of the base pair, RA as a primer is combined with R2C / R2. The complementary strand synthesis using this as a starting point for the synthesis is a reaction up to FA5, Flc at the side terminal. This interaction, the chain synthesis reaction continues, and it is combined with the replacement primer R3 as usual, and the complementary chain synthesis reaction is performed with the replacement of the chain. RA is used as the starting point of the synthesis. The substituted complementary strand at this time is located at the 3 'end of the FA complementary sequence having 5A to the side of R A. In addition, 3 of the single-stranded nucleic acid thus substituted is flanked by the complementary sequence F1 of the same Flc on the same strand, and F1 and F1 coexist in the same molecule can be quickly combined to start the complementary strand synthesis reaction. 3. When the end (F1) is on the same strand, a ring structure containing F2e is formed. The maintenance of the base-pair-possible state of the ring structure wound is shown in Fig. 2 (7). The starting point of the synthesis of the two complementary chains of the oligonucleopeptide FA of the present invention having the F2c = ^ base sequence (7). The mutual structure starting from the cyclic structure is replaced by the reaction product of the synthesis of the complementary chain, and the open end J Jianmei 12 is used as a template to synthesize the complementary chain, and then 3, m possible states. The 3, the ends are located in the same strand

Rlc、、、。5的領域R1的3,末端，依舊是 反應，所以優先進行兩者士人 于内#乂决速 的FA作為模板合成之3,東=二。如此而來，先前說明過 個領域中進行。結果，的反應與同樣反應都在這 交互連結合之核酸一個H:股鏈上互補鹽基序列 換，以其V太她R1 I、個地屬、戈進行互補鍵合成與置 末端R1的η ^綠乍為起點而繼續進行伸長反應。由於3， 下來的反應在3,末端^狀槿 ％狀構造常含有R2e，接 酸（即RA)。 、衣狀構造部份常常是具有R2的寡核苷 的-股K鏈ΪΪ自而繼續進行伸長反應 合成起點而進行互補^ =崢份結合的寡核苷酸作為 雨鏈合成的核酸受到注目，在此處，本Rlc ,,,. The 3, end of the field R1 of 5 is still a reaction, so priority is given to the combination of the FA of the two people in the # 乂 定 速 3, East = 2. As such, it has previously been described in one area. As a result, the reaction and the same reaction are all performed on the H: strand of the interlinked nucleic acid, and the complementary base sequence is replaced by the V ta R1 I, the genus, and the Go to the complementary bond synthesis and η at the terminal R1 ^ Luza continued the elongation reaction as a starting point. Due to the 3, the next reaction is at the 3, and the terminal structure is often composed of R2e, which is connected to the acid (ie, RA). The coat-like structure part is often an oligonucleoside-stranded K chain with R2, so that the elongation reaction is continued and the starting point of the synthesis is complementary. The oligo-nucleotide that is bound to the component has attracted attention as a nucleic acid synthesized by the rain chain. Here, this

第31頁 1221855 五、發明說明（29) 發明一 ^上互補鹽基序列交互連結之核酸也進行合成反 應。也就疋祝，環狀構造開始的互補鏈合成是如圖 2-⑺，RA到達時點時終了反應。接著，依據此核酸合成 之置換核酸互補鏈合成反應開始（圖3 —(8))，不久，此反 應到達冒經作為合成起點的環狀構造部份，於是再度開始 置換反應。如此而纟，由環狀構造開始合成的也被置換， 此結果得到同一股鏈上可以結合的3,末端Rl (圖3 —(1〇))。 此3’末端的R1再與同一股鏈上的R2c結合而開始進行互補 鏈合成反應。而且，此反應的F與1^只是改變稱呼，與圖 2 (7)所發生的疋相同的反應。依據於此，圖所示 的構造是繼續進行本身延長與新核酸生成的新核酸機能。 且圖3- (1 〇 )所示的核酸開始核酸合成反應是與到此為 止所述反應相反的，常以3’末端F1作為合成起點而進行延 長反應。也就是說，本發明中，一個核酸的延長，會伴隨 著供給其他延長反應開始的新核酸，而繼續進行反應。更 進一步’隨著鏈的延長反應，不只是末端，同一鏈上多數 的環狀構造也形成序列。此處環狀構造形成序列是鏈置換 合成反應中鹽基對形成之可能狀態，而募核苔酸的結合， 將提供新核酸生成反應的基礎。不只是末端，鏈的中途也 了以開始合成反應，這樣的組成將達成有效率的增幅反 應。以上作為反向引子的是基於本發明的募核苷酸RA的組 合，延長反應使之伴隨著新核酸的生成。更進_步，本發 明中，此新生成核酸本身的延長，將伴隨更新的核酸的生 成。一連串的反應在理論上是會永久繼續的，而達成極有Page 31 1221855 V. Description of the invention (29) Invention 1 The nucleic acid of the complementary salt sequence on the cross-linking is also synthesized. That is to say, the synthesis of the complementary strand at the beginning of the circular structure is shown in Figure 2-⑺, and the reaction ends when RA reaches the point in time. Then, the synthesis reaction of the complementary nucleic acid complementary strand according to this nucleic acid synthesis was started (Fig. 3-(8)). Soon, the reaction reached the loop structure part which passed through as the starting point of the synthesis, and then started the replacement reaction again. In this way, the synthesis starting from the ring structure is also replaced, and as a result, 3, terminal R1 which can be combined on the same strand is obtained (Fig. 3-(1〇)). The R1 at the 3 'end is then combined with R2c on the same strand to start a complementary strand synthesis reaction. In addition, F and 1 ^ of this reaction only change the name, which is the same reaction as that of 疋 in Figure 2 (7). Based on this, the structure shown in the figure is a new nucleic acid function that continues to extend itself and generate new nucleic acids. In addition, the nucleic acid synthesis reaction shown in Fig. 3- (10) starts the nucleic acid synthesis reaction as opposed to the above-mentioned reaction, and the extension reaction is often carried out using the 3 'end F1 as the starting point of the synthesis. That is, in the present invention, the elongation of one nucleic acid is accompanied by the supply of new nucleic acid to start another elongation reaction, and the reaction continues. Furthermore, as the chain is extended, not only the ends, but also the majority of the cyclic structures on the same chain form a sequence. Here, the loop formation sequence is a possible state for the formation of a salt pair in a strand displacement synthesis reaction, and the combination of ribosomal acid will provide the basis for a new nucleic acid generation reaction. Not only the end, but also the middle of the chain to start the synthesis reaction. Such a composition will achieve an efficient amplification reaction. The above-mentioned reverse primers are based on the combination of the nucleotide-raising RA of the present invention, and the reaction is extended so that new nucleic acids are generated. Further, in the present invention, the extension of the newly generated nucleic acid itself will be accompanied by the generation of newer nucleic acid. A series of reactions will theoretically continue forever, and reach extremely

第32頁 1221855 五、發明說明（30) 效率的核酸增幅。另外本發明的反應是在等溫條件下也 以進行的。 此時蓄積的反應生成物是具有F 1 -R1間鹽基序列及其 互補序列交互連結的構造。正確反覆單位構造的序列兩端 是F2-Fl(F2c-Flc)，再來是R2-Rl(R2c-Rlc)的鹽基序列所 構成領域的連續。例如圖3 —(9)是從5，側開始以 (R2^F2c)-(F1-R2c)-(Rl-Flc)-(F2-R2c)如此順序的連結 狀悲。這是基於本發明的增幅反應，以寡核替酸 起點，由F2(還有R2)開始進行，繼續以本身3甘= 成起點，由F1 (還有R1 )開始的互補鏈合成反應而進行延: 反應’以此原理而進行的反應。 而且，此處最佳狀態之環狀構造部份結合的募桉 是採用本發明募核苷酸FA及RA。然而本發明的核酸;^Page 32 1221855 V. Description of the invention (30) Efficiency of nucleic acid increase. In addition, the reaction of the present invention can be carried out under isothermal conditions. The reaction products accumulated at this time have a structure in which the base sequence between F 1 -R 1 and its complementary sequence are mutually connected. The sequence of the correct iterative unit construction ends at the ends of the sequence consisting of F2-Fl (F2c-Flc), followed by the base sequence of R2-Rl (R2c-Rlc). For example, in Figure 3— (9), the connection from (5) to (R2 ^ F2c)-(F1-R2c)-(Rl-Flc)-(F2-R2c) in this order is sad. This is based on the amplification reaction of the present invention, starting from the oligonucleotide starting from F2 (and R2), and continuing with the complementary chain synthesis reaction starting from F1 (and R1) starting from F3 (and R1). Delay: A reaction based on this principle. In addition, in the optimal state, the loop structure portion is combined with the nucleotides FA and RA of the present invention. However, the nucleic acid of the present invention; ^

應不只限於具有此處構造的寡核苷酸，光是利用由^ J 造開始互補鏈合成反應的寡核苷酸也可以實施。也就σ 說，繼續延長的3,末端若只是環狀構造開始互補鏈^ ^ 置換反應，環狀構造部份可再度適用。環狀構造部^私 =互補鏈合成常是由一股鏈上互補鹽基序列交互連二° 酸作為模板而開始，本發明目的核酸的合成自缺可^核 正確的說，此處合成的核酸是置換後形成的環;^ ^ 過互補鏈合成反應而得到的，若不具有能進行之 = 需環狀構造的3’末端，就沒有了作為新模板的機能。^ 此說，與以FA或是RA開始合成的核酸若改變了，就盔^據 成所期待的指數增幅。由於這個理由，呈二法達 >、有和RA這樣構It should not only be limited to the oligonucleotide having the structure here, but it can also be implemented by using only oligonucleotides starting from the complementary strand synthesis reaction. That is to say, if the extended 3, the end is only a circular structure to start the complementary chain ^ ^ substitution reaction, the circular structure part can be applied again. The circular structural part ^ private = complementary strand synthesis often starts with the complementary base sequence on a strand alternately connecting two acids as a template. The synthesis of the target nucleic acid of the present invention is self-deficient. Nucleic acid is a loop formed after replacement; ^ ^ It is obtained through the complementary strand synthesis reaction. If it does not have a 3 'end that requires a circular structure, it will not function as a new template. ^ This means that if the nucleic acid starting with FA or RA is changed, the helmet ^ is based on the exponential increase expected. For this reason, the two Fada >, You and RA are constructed

第33頁 1221855 案號 89106688 五、發明說明（31) 造的寡核苷酸才對於基於本發明的高度效遂& 用。 千核酸合成有 -連串反應對於作為模板的單股鏈核酸而t，加入以 下的成份’對構成FA及RA鹽基序列互補的鹽基序以妒 成較安定的鹽基對結合’而能在維持酵素活性的溫度中進 行反應。 ’孤又 • 4種寡核苷酸： F A、 RA > 外引子F3、 及外引子R3、 進行鏈置換型互補鏈合成反應的DNa聚合酶、 作為DNA基質的核苷酸。 口 要。ΐΐί此，像PCR過程所需的溫度循環在此處將不需 :寡二ί所謂安定的鹽基對結合是代表存在反應系統中 來所期一條與互補鍵合成起點μ的狀態。可帶 (Tm)以下女疋鹽基對結合的條件例如設定在融解溫度 列一般融解溫度（ΤΐΠ)是5〇%具有互相互補鹽基序 匕= = 結合的狀態時的溫度。設定在融解 度人成H 、’非本發明必要條件，而是考慮到能達成高 鍵；項。作為模板的核酸為雙股 前=進:-此::::好熱二進行，這在反應開始前的 第34頁 2125.3116-PFl.ptc 1221855Page 33 1221855 Case No. 89106688 V. Description of the invention (31) The oligonucleotides produced by the invention are only useful for high efficiency based on the present invention. Thousands of nucleic acids are synthesized-a series of reactions. For single-stranded nucleic acid as a template, t, the following components are added: 'Salt motifs that are complementary to the FA and RA base sequences are combined to form a more stable base pair.' The reaction is performed at a temperature that maintains enzyme activity. Orphan • 4 types of oligonucleotides: F A, RA > Outer primer F3, and outer primer R3, DNa polymerase that performs strand displacement type complementary strand synthesis reaction, and a nucleotide as a DNA substrate. Mouth wants. This means that the temperature cycling required for the PCR process will not be needed here: the so-called stable combination of the salt-based pair represents the state in the reaction system where the starting point μ is synthesized with the complementary bond. For example, the conditions for the bonding of the bases of the son-in-law with a band (Tm) or lower can be set at, for example, the melting temperature, and the general melting temperature (TΐΠ) is 50% of the temperature at the time of having a mutually complementary salt motif. It is not necessary for the invention to set H, ′ in the melting degree, but it is considered that a high bond can be achieved. The nucleic acid used as a template is double-stranded. Pre-in: -this ::::: Good heat two, this is before the reaction starts. Page 34 2125.3116-PFl.ptc 1221855

了酸去總^疋需要維持適合酵素反應之pH值的緩衝劑、為 漸I、二女媒活性之維持及結合時所必需的鹽類、酵素保護 知=有相應必要融解溫度（Tm)的調整劑等共存之下才能In order to remove the total acid, it is necessary to maintain a buffer suitable for the pH of the enzyme reaction, and the salts and enzyme protection necessary to maintain and combine the activity of the female and female mediators = the corresponding necessary melting temperature (Tm) Conditioners and other coexistence

驗ί之螻Ϊ為緩衝劑者常採用如Tris —HC1等具有中性至弱 廡敕、、、作用的緩衝劑。pH依所採用的DNA聚合酶而相 = 類者有K1侧、⑽4W 旦 、、/、核酸融解溫度（Tm)之調整而添加適宜的用 素保護劑者有採用牛血清白蛋白（BSA)及糖類 πΜςΓη β嘁解溫度（Tm)的調整劑一般採用二甲基亞楓 兑曱醯胺（formamide)。使用融解溫度（Tm)調整劑 :述寡核苷酸結合所限制的溫度條件之下而調整即 四烷 碱(Μ" —，N，N，N-trimethylglycine)及 凡土 1 ammonium)對等穩定 (:s:s:a:lilze)作用之鏈置換效率的提升有效。甜菜碱通 i明椤；ί*中添加°.2_3.〇M，以〇.5_ι._度的添加對本 反應的促進作用較佳。此處所提到的融解溫 融：溫度是採向較低溫方向的作用，也必 ί 。辰度及反應溫度等其他條件，依經驗而設定適切 的嚴可度（stringency)與適合反應性的條件。 ㈣ίΐ明t，一連串反應常維持在多種領域之位置關係 的狀怨進行是报重要的特徵。對此特徵而言，要有效率地 防止伴隨非特異性互補*合成而μ的 ;就是說，例如若是某處會發生非特異性反應，就要降低 對於此生成物之後的增幅工程可㊣需要的㈣。且所謂對 1221855 五、發明說明（33) 更多領域控制其反應進行’就是要自由構成對可能類似的 鹽基序列嚴密識別的檢出系統。Testers who use buffers such as Tris-HC1 often use neutral to weak buffers. The pH depends on the DNA polymerase used. Those who have K1 side, ⑽4W denier, and / or nucleic acid melting temperature (Tm) can be adjusted, and those who add suitable protection agents can use bovine serum albumin (BSA) and The regulator of the saccharide πΜς Γη β decomposition temperature (Tm) is generally dimethylformamide. Use the melting temperature (Tm) adjuster: adjust under the temperature conditions restricted by the binding of the oligonucleotide, namely tetramethylene base (M "-, N, N, N-trimethylglycine) and mortal earth 1 ammonium) Equivalent stability (: s: s: a: lilze) The effect of chain replacement efficiency is effective. Betaine is usually added to ° .2_3.〇M, and the addition at a degree of 0.5_ι._ degree can promote the reaction better. The melting temperature mentioned here: The temperature is a function of taking the temperature towards the lower temperature, and it must be too. For other conditions, such as the temperature and reaction temperature, appropriate stringency and reactivity conditions should be set according to experience. It is important to note that a series of reactions that often maintain positional relationships in multiple areas is an important feature of reporting. For this feature, it is necessary to efficiently prevent μ with non-specific complementary * synthesis; that is, for example, if a non-specific reaction occurs somewhere, it is necessary to reduce the need for the subsequent amplification process of this product. ㈣. And the so-called 1221855 V. Invention Description (33) More fields control its reaction ’is to freely construct a detection system that can closely identify possible similar base sequences.

此特徵可以利用遺傳子變異檢出。本發明中所用的外 引子共有4種，2種外引子，2種本發明之募核苷酸而來的 引子。即是，若含有的4種引子不被設計成通用於6個領 域’則本發明的合成反應將無法進行。特別是作為互補鏈 合成起點的各寡核杳酸之3 ’末端，及作為互補序列合成起 點之X1 c領域5 ’末端的序列最為重要。在此處若是檢出設 計上編碼這些重要序列的變異，就要觀察依本發明而得的 合成反應生成物有沒有鹽基缺失或***等變異，或是要作 SfPs之類的遺傳子多型性綜合分析。更具體的說，預測有 變異或夕型性的鹽基就要设計在相當於互補鍵合成起點寡 核普酸的3 ’末端附近（作為互補鏈起點時為5，末端附近）。 若互補鏈合成起點的3,末端及其附近發生錯誤配對 (mismatch)的情形時，將會阻礙核酸互補鏈合成反應。本 發明中’對反應初期生成物末端構造若不能進行反覆反應 時’就無法達成高度增幅的反應。依據於此，例如進行之 反應有錯誤的合成時，構成增幅反應互補鏈合成的各個階 ^又將會文到妨礙而使含有錯誤配對的序列不發生高度之增 幅。結果有效的抑制了錯誤配對之增幅反應，最終將得到 正確的^果。總之基於本發明的核酸增幅反應可以說是具 備有較同度鹽基序列的檢查（check )系統。此特徵對於如 單純2個領域進行增幅反應等PCR方法而言是難以期待的優 點0This feature can be detected using genetic variation. There are a total of four types of primers used in the present invention, two types of primers, and two types of primers derived from the nucleotides of the present invention. That is, if the four types of primers contained are not designed to be used in six areas' in general, the synthesis reaction of the present invention will not proceed. In particular, the 3 'end of each oligonucleotide as the starting point for the synthesis of the complementary strand and the sequence at the 5' end of the X1 c domain as the starting point for the synthesis of the complementary sequence are the most important. Here, if mutations encoding these important sequences are detected in the design, it is necessary to observe whether the synthetic reaction product obtained according to the present invention has mutations such as base deletion or insertion, or to be used for genetic subtypes such as SfPs. Comprehensive analysis. More specifically, it is predicted that a base having a mutation or a base type should be designed near the 3 'end of the oligonucleotide corresponding to the starting point of the complementary bond synthesis (5 as the starting point of the complementary strand, and near the end). If mismatch occurs at the 3, end of the complementary strand synthesis starting point and its vicinity, the nucleic acid complementary strand synthesis reaction will be hindered. In the present invention, "if the terminal structure of the product at the initial stage of the reaction cannot be repeatedly reacted", a highly amplified reaction cannot be achieved. Based on this, for example, when the reaction is carried out with an erroneous synthesis, each stage of the complementary chain synthesis that constitutes the amplification reaction will be hindered so that the sequence containing the mismatch does not increase in height. As a result, the amplification response of the wrong pairing is effectively suppressed, and finally the correct result will be obtained. In short, the nucleic acid amplification reaction based on the present invention can be said to have a check system having a relatively homologous base sequence. This feature is an unanticipated advantage for PCR methods such as simple amplification in two areas.

第36頁 I221855Page 36 I221855

始人明中所採用募核苷酸的特徵領域Xlc是開 狀二二之生成時，本寡核苷酸將不形成環 構k 依據於此，發生引JTL ^ S/& db Ati m 卜M紐、i +、+ 刟子雙體之非特異增幅得到原理 上的解決方法，而對反應特異性之提高有所貢獻。 引子：ϋΓ月中，F3(圖卜(2))趟(圖2-(5))所示的外 引子組合將可以㈣溫條件下進行上述的一$串反岸。也When Xlc is a characteristic field of nucleotide collection used in Shirenming, the oligo will not form a loop structure based on this. JTL ^ S / & db Ati m The non-specific increase of M button, i +, and zongzi dimers has been solved in principle, and has contributed to the improvement of reaction specificity. Introducer: In the middle of the month, the outer primer combination shown in F3 (Figure (2)) (Figure 2- (5)) will be able to perform the above-mentioned one-string anti-shore under the condition of high temperature. and also

就是說本發明提供了含有前述[9]所示工程一股鏈上1補 鹽基序列交互連結之核酸增幅方法。此方法是選擇f2c/f2 間、R2C/R2間、Flc/F1間及Rlc/R1間能發生安定的結合的 溫度條件，且希望此設計有助於在F3c/F3間及r3c/r3間分 別與F2c/F2間及R2C/R2間以連續堆疊現象結合。 本發明中採用核酸合成及增幅之用語。在本發明中核 酸合成是代表由作為合成起點的寡核苷酸開始進彳^核酸延 長反應的意思。而當合成增加，再加上其他核酸的生成， 此生成核酸之延長反應連續進行時’ 一連串反應的總合就 稱為增幅。 一股鏈上一部份的F 1 c 同一股鏈上之Flc結That is to say, the present invention provides a nucleic acid amplification method comprising the interlinking of a complementary base sequence on a strand of the project shown in the aforementioned [9]. This method is to select the temperature conditions for stable binding between f2c / f2, R2C / R2, Flc / F1, and Rlc / R1, and hope that this design will help to distinguish between F3c / F3 and r3c / r3. Combined with F2c / F2 and R2C / R2 in a continuous stacking phenomenon. In the present invention, the terms of nucleic acid synthesis and amplification are used. In the present invention, the meaning of nucleic acid synthesis means that the nucleic acid extension reaction starts from the oligonucleotide which is the starting point of synthesis. When the synthesis is increased and the production of other nucleic acids is added, the extended reaction of the generated nucleic acid is continuously performed. The sum of the series of reactions is called the increase. A part of F 1 c on one strand of chain

其次，具備有在3’末端可與同 結合之領域F1，而此領域F1因可與 合’使其可形成含有鹽基對結合可能之領域F2c的單股鏈 核酸是本發明重要的構成要素。這樣的單股鏈核酸基於以 下所述的原理而提供此功能。也就是說，基於持有以下將Secondly, it is provided with a domain F1 which can bind to the same at the 3 ′ end, and since this domain F1 can be combined, it can form a single-stranded nucleic acid containing a domain F2c containing a base pair that can bind, which is an important component of the present invention. . Such single-stranded nucleic acids provide this function based on the principles described below. That is, based on holding the following

1221855 五'發明說明（35) 述及之結構的引子將可進行互補鏈合成反應。 5，-[與 位於引子内領域Xlc可結合的領域XI ]—[鹽基對結合形成環 狀構造狀態之可能序列]-[領域XI c ]-[具有與模板互補序 列的領域]-3’The primers of the structure mentioned in 1221855 Five 'Invention Note (35) will be able to perform complementary strand synthesis reactions. 5,-[Domain XI that can be combined with domain Xlc located in the primer] — [Possible sequence for the combination of base pairs to form a circular structure state]-[Domain XI c]-[Domain with sequence complementary to the template] -3 ’

具有與模板互補序列的領域是代表與F 1互補鹽基序列 (引子-FA)及與Rlc互補鹽基序列（引子—ra)兩種序列之 意。且此時構成可合成核酸的鹽基序列是含有由領域F丨至 領域R1 c的鹽基序列，及由具有與此鹽基序列互補鹽基序 列的領域R1至Flc的鹽基序列。一方面，引子内部可結合 的XI c與XI可以是任何序列。的確，引子—Fa與ra之間較希 2疋與領域Xlc/Xl之序列不同的序列。A field having a sequence complementary to the template is intended to represent two sequences complementary to the F 1 complementary base sequence (primer-FA) and to the Rlc complementary base sequence (primer-ra). In this case, the base sequence constituting the synthesizable nucleic acid is a base sequence including a domain F1 to a domain R1 c, and a base sequence having a domain base sequence complementary to the base sequence. On the one hand, the XI c and XI that can be combined inside the primer can be any sequence. Indeed, the primers—Fa and Ra—are sequences that differ from the sequences in the domain Xlc / Xl.

首先，模板核酸的領域F1開始與前述的引子—FA進行 互補鏈合成反應。接著，合成出來之互補鏈的領域R丨c形 成鹽基對結合的可能狀態，此處一部份的引子將與之結合 而作為互補鏈合成反應的起點。此時，由於合成之互補鏈 3 ’末端是具有與構成最初合成鏈5，末端部份之引子— fa的 互補鹽基序列，在3 ’末端持有領域X丨，所以此處將與同一 股鏈上之領域X1 c結合而同時形成環狀構造。如此而來， 前述本發明的特徵是提供3，末端構造，之後的反應將在開 始的反應系統中顯示出最期待的狀態。且此時與環狀構造 結合的寡核脊酸在3，末端具有環狀構造内之領域X2c互補 的領域X2，及在5,侧有領域X1。先前反應系統將在可合成 使用引子-FA及RA作為模板核酸之互補鏈核酸的3，末端帶 來環狀構造。此方法以較短的引子有效的提供本發明特徵First, the domain F1 of the template nucleic acid starts a complementary strand synthesis reaction with the aforementioned primer-FA. Then, the domain of the synthesized complementary strand R 丨 forms a possible state of binding of the base pair, and a part of the primers here will be combined with it as a starting point for the synthesis reaction of the complementary strand. At this time, since the 3 ′ end of the synthetic complementary strand has a complementary base sequence with the primer-fa, which constitutes the end portion of the first synthetic strand 5, and holds the domain X 丨 at the 3 ′ end, it will be the same strand here The domain X1 c on the chain combines to form a ring structure at the same time. In this way, the aforementioned feature of the present invention is to provide a 3, terminal structure, and subsequent reactions will show the most anticipated state in the initial reaction system. At this time, the oligonucleo spine which is bound to the cyclic structure is at 3, and the end has the field X2 complementary to the field X2c in the cyclic structure, and the field X1 is at the 5 side. In the previous reaction system, the 3 'end of the complementary strand nucleic acid which can synthesize the primer-FA and RA as a template nucleic acid has a circular structure. This method effectively provides the features of the present invention with shorter primers.

第38頁 1221855 五、發明說明（36) 的末端構造。一方面，本狀態中，首次提供以構成環狀構 造的鹽基序列全體作為引子，比較長引子的合成更必要。 利用在反向引子含有限制酶認識領域的鹽基序列，本 發明構成較特異的狀態。基於圖6，在此對反向引子含有 限制酶認識領域的方法做較具體的說明。當圖6 - ( d )完成 時’編碼於反向引子内含有的限制酶認識部位的限制酶將 作用而造成缺口（nick)。此缺口作為合成起點而開始鏈置 換型的互補鏈合成反應。由於反向引子是位於構成（D)之 雙股鏈核酸的兩端，互補鏈合成反應也是由兩端開始進 行。基本上’基於早先所記載之技術以SDA法的原理，作 為模板的鹽基序列與本發明中互補鹽基序列交互連結之構 ^ 而構成本發明特有的核酸合成系統。且反向引子互補 鍵加入缺口的部份是不得不設計成取回不發生限制酶切斷 雙股鏈這樣的核酸酶耐性的dNTp誘導體。 在反向引子也可以***RNA聚合酶的啟動子。此情形 與先前應用SDA法的狀態相同，由圖6 —(D)的兩端開始以認 識此啟動子的RNA聚合酶進行轉錄的工程。 以本發明合成的核酸是由所謂單股鏈互補鹽基序列開 始而構成的’所以形成的大部分是鹽基對結合的狀態。利 用此特徵，將可檢出合成生成物。在溴化乙錠（ethfdium bromide)、SYBR Green I、或是 Pico (jreen 之類對雙股鏈 特異的嵌合（interchalate)螢光色素存在下，實施本發明 的核酸合成方法，伴隨著生成物的增加，觀察螢光強^增 大的情形。如此監控，冑可以追蹤閉鎖系統中真實合：丄Page 38 1221855 V. Terminal structure of invention description (36). On the one hand, in this state, for the first time, the entire base sequence constituting the loop structure is provided as a primer, and synthesis of a longer primer is more necessary. The present invention constitutes a more specific state using a base sequence in the field of recognition of a restriction enzyme in a reverse primer. Based on Fig. 6, a method for identifying a restriction enzyme in a reverse primer will be specifically described. When Figure 6-(d) is completed, the restriction enzyme that encodes the recognition site for the restriction enzyme contained in the reverse primer will act and cause a nick. This gap serves as a starting point for synthesis and starts the complementary strand synthesis reaction of the strand displacement type. Since the reverse primer is located at both ends of the double-stranded nucleic acid constituting (D), the complementary strand synthesis reaction also starts from both ends. Basically, based on the technology described earlier and based on the principle of the SDA method, the structure in which the base sequence used as a template and the complementary base sequence in the present invention are mutually linked ^ constitutes a nucleic acid synthesis system unique to the present invention. In addition, the part where the complementary bond of the reverse primer is added to the gap has to be designed to retrieve a nuclease-resistant dNTp inducer that does not undergo double-strand cuts by restriction enzymes. An RNA polymerase promoter can also be inserted into the reverse primer. This situation is the same as that of the previous application of the SDA method, and the transcription engineering is started from the ends of Fig. 6- (D) with the RNA polymerase that recognizes this promoter. The nucleic acid synthesized by the present invention is composed of a so-called single-stranded complementary salt-based sequence. Therefore, most of the formed nucleic acids are in a state where the salt-base pair is bound. With this feature, synthetic products can be detected. The nucleic acid synthesis method of the present invention is performed in the presence of ethfdium bromide, SYBR Green I, or Pico (jreen) specific interchain fluorescein, and the product is accompanied by the product. Increase, observe the increase in fluorescence intensity ^. With this monitoring, 胄 can track the true combination in the locking system: 丄

12218551221855

五、發明說明（37) 應的時間（real time)。這種檢出系統也有考慮過應用於 PCR法，但由於與引子雙體等等訊息（signal)的發生並沒 有區別的方法，所以問題彳艮多。然而應用在本發明的情形 下，由於非特異性鹽基對結合增加的可能性非常低，將 期待同時得到兩敏感度且少雜訊（n 〇 i s e)的結果。與雙股 鏈特異的嵌合劑同樣的，作為實現均一系之檢出系統的方 法將可以利用螢光能量的轉移。 可支援本發明核酸合成方法的還有作為鏈置換型互補 鏈合成反應觸媒的DNA聚合酶。上述反應中，不一定要含 有需要鏈置換型聚合酶的反應步驟。然而，構成試藥的單 純化，且以經濟的觀點考量，只利用一種DNA聚合酶將較 為有利。此種DNA聚合酶已知具有以下所述的特色。還 有’這裡所k到酵素的各種變異體限於具有序列依存性互 補鏈合成活性與鏈置換活性者，而在本發明中使用。此處 所提到的變異體是將酵素中具有必要觸媒活性的構造特^ 取出，或是經如胺基酸變異等而得，顯示其觸媒活性、安 定性、或是耐熱性被改變者。5. Description of the invention (37) Real time. This detection system is also considered to be applied to the PCR method, but there are many problems because there is no difference between the method and the occurrence of signals such as primer duplexes. However, in the case of application of the present invention, since the possibility of non-specific base-pair increase binding is very low, it is expected that a result of both sensitivity and low noise (n oi s e) will be obtained at the same time. As a double-strand specific chimer, as a method for achieving a homogeneous detection system, it is possible to utilize the transfer of fluorescent energy. A DNA polymerase which is a catalyst for the strand displacement type complementary strand synthesis reaction can also be used to support the nucleic acid synthesis method of the present invention. The above reaction does not necessarily include a reaction step requiring a strand displacement polymerase. However, it is advantageous to use only one DNA polymerase for the simplification of the reagent and from an economic point of view. Such a DNA polymerase is known to have the characteristics described below. Various variants of the enzymes described herein are limited to those having sequence-dependent complementary chain synthesis activity and strand displacement activity, and are used in the present invention. The variants mentioned here are those that have the necessary catalytic activity in the enzyme, or are obtained by mutations such as amino acids, which show that their catalytic activity, stability, or heat resistance has been changed. By.

Bst DNA 聚合酶Bst DNA polymerase

Bca (exo-）DNA 聚合酶DNA 聚合酶的Klenow 片段Vent DNA 聚合酶Bca (exo-) DNA polymerase Klenow fragment of DNA polymerase Vent DNA polymerase

Vent (exo-) DNA聚合酶（Vent DNA聚合酶中，去除外核酸 酶（exonuclease)活性者）Vent (exo-) DNA polymerase (Vent DNA polymerase removes exonuclease activity)

DeepVent DNA 聚合酶DeepVent DNA polymerase

DeepVent (exo-) DNA 聚合酶（DeepVent DNA 聚合酶去除外DeepVent (exo-) DNA polymerase (DeepVent DNA polymerase removes

第40頁 1221855 五、發明說明（38) 核酸酶（e X ο n u c 1 e a s e )活性者） Φ29噬菌體（phage) DNA聚合酶 MS-2噬菌體（phage) dna聚合酶 Z-Taq DNA聚合酶（寶酒造） KOD DNA聚合酶（東洋紡績） MU取此人處所列舉的酵素中，Bst DNA聚合酶及Bca (exo_) DNA/ δ酶是特別具有某種程度的耐熱性，且觸媒活性也 Ϊ n i人期待的酵素。本發明的反應較希望的狀態是在 條件下實施，為了融解溫度（Tm)的調整等問題，不一 2 Γ要Γ用適合酵素安定性的溫度條件。依據於此，酵 摇批：t:一項期待中的條件。此外，雖說是等溫反應， 射鈦，ι! ί ϊ初模板的核酸仍可能需要進行加熱變性，因此 声…、，酵素的利用在試驗（assay)流程中選擇的領域較 Μ 0 丨π2ΐί(ΕΧ〇_)卵4聚合酶是同時具備有鏈置換活性及高 二互補鏈ΚΙ雍1此已知以ΜΑ聚合酶進行伴有鏈置換 strand 絲加單股鏈結合蛋白（如16 rl. π mg Pr〇tein)會促進反應的進行（PaUl M. 1 998^ ? NatUre Genetics 19， 225-232, July, )。在本發明中應用這項作用，添加單股鏈社合蛋白 DNA聚合|母而言，作為單股鏈姓 32很有效。 F勹早奴鍵、、口。蛋白的T4 gene 尚且，已知不具有3，—5,外核酸酶（3、5，Page 40 1221855 V. Explanation of the invention (38) Nuclease (e X ο nuc 1 ease) active person Φ29 phage (phage) DNA polymerase MS-2 phage (phage) dna polymerase Z-Taq DNA polymerase (Takara Shuzo ) KOD DNA polymerase (Toyobo) MU. Among the enzymes listed by this person, Bst DNA polymerase and Bca (exo_) DNA / δ enzyme are particularly heat-resistant, and their catalytic activity is also high. Looking forward to enzymes. The reaction of the present invention is preferably performed under conditions. In order to solve problems such as the adjustment of the melting temperature (Tm), the temperature conditions suitable for enzyme stability are required. Based on this, leavened batch: t: an expected condition. In addition, although it is an isothermal reaction, Titanium injection, ι! Ί The nucleic acid of the initial template may still need to be denatured by heating. Therefore, the use of enzymes in the assay process is more selective than Μ 0 丨 π2ΐί ( Εχ〇_) egg 4 polymerase has both chain displacement activity and high-secondary complementary chain ΙΙΙ. It is known that MAP polymerase is used to perform strand replacement with strand replacement and single-stranded binding protein (such as 16 rl. Π mg Pr 〇tein) will promote the reaction (PaUl M. 1 998 ^? NatUre Genetics 19, 225-232, July,). In the present invention, when this function is applied, the single-stranded cosynthetic protein DNA polymerization | parent is effective as the single-stranded surname 32. F 勹 early slave key ,, mouth. The T4 gene of the protein is also known not to have 3, -5, exonuclease (3, 5,

HI 第41頁 1221855 五、發明說明（39) -----HI Page 41 1221855 V. Description of Invention (39) -----

exonuclease)活性的DNA聚合酶會有在進行互補鏈人成到 達，板的5’末端部份時不停止，而多合成丨個鹽基二出之 狀態的情形。本發明由於在互補鏈合成到達末端時的3，末 端序j接著就是下一個互補鏈合成的開始，所以可以避免 發生前述的現象。然而，DNA聚合酶在3,末端處之核酸的 附加在經驗上通常是A。因此，為了避免dATp錯誤地附加i 鹽基的問題，3’末端開始的合成若是能選擇以八開始的序 列較佳。此外，互補鏈合成時，若3,末端有突出，將會利 用3’-5’外核酸酶的活性使該處消化成盲端（blunt end)。 例如，由於天然型的Vent DNA聚合酶具有此活性，與Vent (Εχο·>) DNA聚合酶混合使用，將可以避免這項問題。exonuclease) active DNA polymerase may generate complementary strands without stopping at the 5'-end portion of the plate and synthesize more than one base. In the present invention, since 3 when the complementary strand synthesis reaches the end, the end sequence j is then the start of the next complementary strand synthesis, so the aforementioned phenomenon can be avoided. However, the addition of a nucleic acid at the 3 'end of a DNA polymerase is usually empirically A. Therefore, in order to avoid the problem that dATp erroneously attaches the i salt group, it is better to select a sequence starting from eight at the 3 'end. In addition, if the 3, terminus of the complementary strand is projected, the 3'-5 'exonuclease activity will be used to digest it into a blunt end. For example, because natural Vent DNA polymerase has this activity, it can be avoided by mixing with Vent (Eχο ·) DNA polymerase.

、本發明的核酸合成方法，或是對增幅方法必要的各種 试藥可以由事先包裝好的套組提供。具體的說，由本發明 可提供以互補鏈合成引子、或是作為置換用外引子必要的 各種寡核苷酸、作為互補鏈合成基質的dNTp、進行鏈置換 型互補鏈合成的DNA聚合酶、適合酵素反應的緩衝液、還 有編碼必要合成反應生成物檢出的必要試藥等構成的套 組。特別是，本發明較希望的狀態是希望在反應中途不添 加试藥’所以供給已分裝好1次反應必要試藥的反應容 器’只要添加樣本就可以開始反應的狀態。利用發光訊息 及螢光訊息，在反應容器中就可以進行反應生成物檢出的 系統，將可以全面廢止反應後容器的開封。這是在污染防 止上非常期待的進步。 其次’以本發明合成、單股鏈上互補鹽基序列相互連The nucleic acid synthesis method of the present invention or various reagents necessary for the amplification method may be provided from a packaged package. Specifically, according to the present invention, various oligonucleotides necessary for synthesizing primers with complementary strands or external primers for replacement, dNTp as a substrate for complementary strand synthesis, and DNA polymerases for performing strand displacement complementary strand synthesis can be provided. A set of enzyme reaction buffers and necessary reagents for detecting the necessary synthetic reaction products. In particular, a more desirable state of the present invention is a state in which it is desired not to add a reagent in the middle of the reaction, so that a reaction container that has been dispensed with a reagent necessary for the reaction once is supplied, and the reaction can be started by adding a sample. The system that can detect reaction products in the reaction container by using the luminous and fluorescent information will completely abolish the unsealing of the container after the reaction. This is a highly anticipated progress in pollution prevention. Secondly, the present invention synthesizes and interconnects complementary base sequences on a single strand.

第42頁 1221855 五、發明說明（40) m:以下述及其實用性。第―，伴有互補 ，備於…刀子内之特殊構造優點^舌用。此特徵將可 ::檢义較容易。也就是說隨著與互補鹽基序列的蹿二 :結合，自已知訊息的增減進行核酸檢出的系統。例：工 :所=及，雙股鏈特異嵌合劑作為檢出劑的方 二貫現在本發明生成物特徵出現時檢出的系統。這 樣的檢出糸統中，本發明的合成生成物經過一次加= 溫度時生分子内結合的速度將會優先於 基對結合。—方面，若是非特異反應 性合使2子八子以上於、不雜具有乂種分子内互補序列，故經熱變 : 無法立刻回復原來的雙股鏈。 此而來，檢出珂再追加加熱變性工程，伴 應的干擾將減輕。使用不且有 、/、ι±反 ,^ ^ 從用个八有對熱耐性的DNA聚合酶時， 工程也具有反應停止的意義，對 制廷一點而言也是有利的。 了』幻役 造。鹽基對結合可能狀態的環狀構 =了： ！可此狀恶的環狀構造顯示於圖4。由圖4可 了％，％狀構造是由介於引子結合 F2c(X2c)與F2c_Flc(xlc)之 =^ 二基序列 F2c-F1c 間（一般以X2c — X1c 間顯 ^ ^ ^ ^ ^ ^ ^ 鹽基序列。依據於此，“具有編石：=由板板而來的 針作雜交，將可進行對模柄?於此領域互補序列的探 乂將了切對模板有特異性的檢出。此外 域：疋鹽基對結合可能狀態之區$ 、 加熱變性。且構成本發明增幅 ：：在雜乂刖先 阳汉應生成物裱狀構造的鹽基Page 42 1221855 V. Description of the invention (40) m: The following and its practicality. No. ——, accompanied by complementary, prepared for the special structural advantages of the knife. This feature will be easier :: forensics. That is, a system for detecting nucleic acids from the increase or decrease of a known message by combining with the second base of a complementary base sequence. Example: Work: So, the double-strand-specific chimeric agent is used as the detection agent. The system is now detected when the characteristics of the product of the present invention appear. In such a detection system, when the synthetic product of the present invention undergoes a single temperature increase, the rate of in-molecule binding will take precedence over the base-pair binding. -On the other hand, if the non-specific reactivity makes 2 or more sons and 8 sons or more, there is no intramolecular complementary sequence, so after thermal change: the original double strand cannot be recovered immediately. As a result, additional heat denaturation process will be added to detect Ke, and the interference will be reduced. The use of a non-, /, ι ±, ^ ^, ^ ^ From the use of a DNA polymerase that has heat resistance, engineering also has the meaning of stopping the reaction, which is also beneficial to the system. It's a fantasy. The cyclic configuration of the possible state of the base pair binding: =! The ring structure which can be like this is shown in FIG. 4. As shown in Fig. 4, the% and% -like structures are composed of the primers that bind between F2c (X2c) and F2c_Flc (xlc) = ^ The two-base sequence F2c-F1c (usually marked by X2c-X1c) ^ ^ ^ ^ ^ ^ ^ Based on this, "have a lithograph: = needles from plate to plate hybridization, will be able to carry out the detection of the complementary handle of the mold handle in this field will cut the specific detection of the template. Outside domain: the region where the hydrazone base pair can be combined, heat denaturation, and constitute the increase of the present invention: the basal structure of the mounting structure of the product in the pre-Han hybrid

第43頁 1221855 五、發明說明（41) 序列可以是任何長度。所以，以探針雜交作為目的的情形 下，引子所結合的領域與探針雜交的領域分別進行，所/ 構成了避免兩者競爭的理想反應條件。 本發明所期望的狀態是得到佔多數單股核酸鏈上鹽 對結合的環狀構造。這代表在核酸一分子上可能有多數^ 針雜交之意’而可得到高敏感度的檢出。此外不只是敏^ 度’例如凝集反應等基於特殊反應原理的核酸檢出方法I 將可行。例如以聚氣乙烯乳劑（P〇l ystyrene latex)之類 微粒子固定的探針加入本發明反應生成物中，伴隨著探針 的雜交’觀察乳劑粒子的凝集。凝集的強度以光學測定， 將了以得到高敏感度且定量的觀察結果。或是，以肉眼觀 察凝集反應，而構成不使用光學測定裝置的反應系統。 而且’可以在1個核酸分子進行相當多數標誌結合的 ^發，反應生成物也可以用色譜分析法檢出。免疫測試的 分野疋利用以肉眼即可檢出的標定進行色譜媒體的分析方 法（免疫色譜分析法），而使之實用化。此方法是基於以色 譜媒體固定的抗體與標定抗體互相連結分析物成為三明▲ (sandwich)構造’再洗去未反應之標定成份的原理。本i 明的反應生成物可以應用這項原理進行核酸分析。也就是 說，編碼環狀構造部份標定的探針經以被色譜媒體固定的 捕捉用探針截流（trap)，再進行色譜媒體中的分析。捕捉 用探針可以利用編碼環狀構造部份的互補序列。本發明的 反應生成物由於大部分伴隨環狀構造，所以多與標定探針 結合，所以可以得到肉眼即可辨識的訊息。Page 43 1221855 V. Description of the Invention (41) The sequence can be of any length. Therefore, in the case of probe hybridization as the purpose, the domain to which the primers bind and the domain to which the probe hybridizes are performed separately, which constitutes an ideal reaction condition to avoid competition between the two. A desirable state of the present invention is to obtain a circular structure in which salt pairs on most single-stranded nucleic acid chains are bound. This means that there may be a large number of needle hybridizations on a nucleic acid molecule, and a highly sensitive detection can be obtained. In addition, not only the sensitivity, such as the agglutination reaction, the nucleic acid detection method I based on a special reaction principle, is feasible. For example, a microparticle-immobilized probe such as polystyrene latex is added to the reaction product of the present invention, and the aggregation of the emulsion particles is observed along with the hybridization of the probe. The intensity of the agglutination was measured optically to obtain a highly sensitive and quantitative observation. Alternatively, the agglutination reaction is observed with the naked eye, and a reaction system is constructed without using an optical measurement device. Furthermore, a considerable number of markers can be bound to one nucleic acid molecule, and the reaction product can also be detected by chromatography. Fumino Aya for immunoassay uses a chromatographic media analysis method (immunochromatographic analysis method) that uses a standard that can be detected with the naked eye to make it practical. This method is based on the principle that the antibody immobilized on the chromatographic medium and the calibration antibody are connected to each other and the analyte becomes a sandwich structure, and then the unreacted calibration components are washed away. The reaction products of the present invention can be used for nucleic acid analysis using this principle. That is to say, the probes calibrated by the coding loop structure are trapped with capture probes fixed by the chromatographic medium, and then analyzed in the chromatographic medium. The capture probe can utilize a complementary sequence encoding a circular structural portion. Since most of the reaction products of the present invention have a ring structure, they are often combined with a calibration probe, so that information that can be recognized by the naked eye can be obtained.

第44頁 1221855 五、發明說明（42) 反應對結合可能領域的本發明 用固定右二他檢出系統。例如’可以是利 (Ρ-：1# —t ，t , ^ ： 狀構造部份之探針以雙成二 更高敏感度的螢光n:;的喪合劑標定，可以進行 成之核酸的3,側盥5,側兩^而蹄可以積極地利用本發明合 構造。例如，—^的環狀播^基對結合可能形成的環狀 鹽基序列部份，另 作為正常型與異常型共通的 領域的鹽基序列4C設計成兩者會生成不同 另-方的領域則確；有探m認基因之存在， 本發明核酸合成反應由於；η:徵的分析系統。 有以一般螢光光度計作真电時條件下進行，所以具 點。到此為止的同一鏈上二=間为析而值得大書特書的優 而’以本發明得到單股鏈：；==已知的。然 是含有可與其他募核苔酸睡序=互連結的核酸 份，在這一點上是新發明=十、、、°&之夕數環狀構造部 一方面，相營於V 士 & 份也可以利用作為探^發明反應生成物的多數環狀構造部 可以集積高密度的探=t如，DNA晶片在有限的範圍内 能有固定募核苷酸的數W而現在技術仍限制一定面積只 物，將可以結合多數的。此，若能利用本發明反應生成 本發明反應生成物將可：2:高密度固定化。A就是說， 有良好效果。反應生成f為探針，固定在DNA晶片上而 生成物在增幅後以已知的方法固定，或Page 44 1221855 V. Description of the invention (42) The invention of the reaction paired with the possible field of use The fixed right binocular detection system. For example, 'may be ligated (P-: 1 # —t, t, ^: the probe of the structure-like part is calibrated with the fluorescent compound n :; which has a higher sensitivity of two to two, and can be used for nucleic acid.) 3, side 5 and side 2 and hooves can actively use the composite structure of the present invention. For example, the ring-shaped base pair of-^ combined with the part of the cyclic base sequence that may be formed, and it is also regarded as a normal type and an abnormal type. The base sequence 4C of the common field is designed so that the two will generate different fields. It is true that there is a probe to recognize the existence of the gene, and the nucleic acid synthesis reaction of the present invention is due to the η: sign analysis system. There is general fluorescence The photometer is performed under the conditions of true electricity, so it is pointy. So far, the same chain on the two chains is worth analyzing and is worthy of a large book and special book, and 'single chain is obtained by the present invention: == known However, it contains nucleic acids that can be linked to other nucleic acid-associated peptides. This is a new invention. At this point, the number of ring structures is equal to V. & parts can also be used as the probe reaction product of most of the circular structure can accumulate high-density probes = t such as DNA The chip can have a fixed number of nucleotides W within a limited range, and the current technology still limits a certain area of material, which can be combined with a majority. Therefore, if the reaction of the present invention can be used to generate the reaction product of the present invention, it can: 2 : High-density immobilization. A means that it has a good effect. The reaction generates f as a probe, which is fixed on the DNA wafer and the product is fixed by a known method after the amplification, or

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是利用固定化的寡核苷酸作為本發明增幅反應的寡核苷 酸，結果得到固定化的反應生成物，這樣採用固定化的探 針，在有限的領域中將可進行多量試料DNA的雜交，期待 可得到高訊息的結果。 B式簡單說明 圖1表示本發明理想狀態的反應原理之部分（丨）—（4)的 模式圖。 圖2表示本發明理想狀態的反應原理之部分（5 ) — ( 7 ) 模式圖。 ' 圖3表示本發明理想狀態的反應原理之部分（$ )— 的模式圖。 圖4表示本發明之單股核酸序列形成環狀構造的模 圖。 、：、 圖5表示以本發明為基礎形態之部分（A ) — ( β )的模 圖〇 、工、 圖圖6表示以本發明為基礎形態之部分（c) —(D)的模式 圖7表示Ml 3mpl8的標的鹽基序列中，構 鹽基序列的位置關係圖。 核甘酸各 圖8表示以M13mP18作為模版，依本發明之 合成方法而得到生成物的瓊脂糖（ r〇s ^鏈核酸 片。 包冰結果照 行1 : XIV標準品標記The immobilized oligonucleotide is used as the oligonucleotide for the amplification reaction of the present invention, and as a result, an immobilized reaction product is obtained. In this way, by using the immobilized probe, a large amount of sample DNA can be hybridized in a limited field. , Expect to get high-information results. Brief Description of Formula B Fig. 1 is a schematic diagram showing part (丨)-(4) of the reaction principle of the ideal state of the present invention. Fig. 2 shows part (5)-(7) schematic diagrams of the reaction principle of the ideal state of the present invention. Fig. 3 is a schematic diagram showing a part ($) of the reaction principle of the ideal state of the present invention. Fig. 4 is a diagram showing a circular structure of a single-stranded nucleic acid sequence of the present invention. Fig. 5 shows a schematic diagram of part (A)-(β) based on the present invention. Fig. 6 shows a schematic diagram of part (c)-(D) based on the present invention. 7 represents a positional relationship diagram of the salt-forming sequence in the target salt-based sequence of Ml 3mpl8. Figure 8 shows agarose (r0s) strand nucleic acid fragments obtained using M13mP18 as a template in accordance with the synthesis method of the present invention. Figure 1 shows the results of ice-packing: XIV standard labeling

!221855! 221855

行2 :1 fm〇l M13mpl8 dsDNA 行3 ·無標的物 圖9表示依實施例1得到本發明妨 较月核酸合成反應的生成 經限制酶消化再以瓊脂糖電泳所得結果照片。 SUPER LADDER-LOW標準品標記 精製物的B a m ΗI消化物 精製物的Pvu I I消化物 精製物的Hindlll消化物 物， 行1 行2 行3 行4 圖1 0表示以Ml 3mpl 8作為模板，添加甜菜碱，得到本 發明單股鏈核酸合成方法的生成物，再經瓊脂糖電泳所得 結果照片。0、0. 5、1、2是代表反應液中添加甜菜碱的濃 度（M)。此外，N代表陰性對照，-21代表模板DNA的濃产 10一21 mol。 又 圖11表示由HBV來的標的鹽基序列中，構成寡核苔酸 的各鹽基序列位置關係圖。 / ^ 圖12表示以組合M13mpl8的HBV- M13mpl8作為模板， 以本發明單股鏈核酸合成方法得到的生成物，再”經遭脂糖 電泳所得結果照片。 '0 行1 : XIV標準品標記 行2 :1 fm〇i HBV-M13mpl8 dsDNA 行3 :無標的物 圖1 3表示以本發明單股鏈核酸合成方法得到沾止上 〜的生成 物’經鹼變性，再以膠體電泳所得結果照片。 行1 : λ Phage的Hindi 11消化斷片Row 2: 1 fm0l M13mpl8 dsDNA Row 3 · No standard substance Figure 9 shows the result of the synthesis of the comparative nucleic acid synthesis reaction of the present invention obtained in Example 1 after restriction enzyme digestion followed by agarose electrophoresis. SUPER LADDER-LOW Standard Marks Refined B am ΗI Digested Refined Pvu II Digested Refined Hindlll Digested, Row 1 Row 2 Row 3 Row 4 Figure 10 shows Ml 3mpl 8 as a template, added Betaine, the product of the method for synthesizing single-stranded nucleic acid of the present invention, and the result photograph obtained by agarose electrophoresis. 0, 0.5, 1, and 2 represent the concentration (M) of betaine added to the reaction solution. In addition, N represents a negative control, and -21 represents a concentration of 10-21 mol of template DNA. Fig. 11 is a diagram showing the positional relationship between the respective base sequences constituting the oligonucleotide in the target base sequence derived from HBV. / ^ Figure 12 shows the photos of the results obtained by lipopolyelectrophoresis using the HBV-M13mpl8 combination of M13mpl8 as a template and the product obtained by the single-stranded nucleic acid synthesis method of the present invention. '0 Row 1: XIV standard labeling row 2: 1 fm〇i HBV-M13mpl8 dsDNA Row 3: No standard substance Figure 13 shows the product obtained by the single-stranded nucleic acid synthesis method according to the present invention, which was denatured with alkali, and then obtained by colloid electrophoresis. Line 1: Hindi 11 digestion section of λ Phage

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行2 ··實施例1的反應生成物 行3 ··實施例3的反應生成物 圖14作為標的的M13mpl8濃度改變時，以太1 ♦發明單月凡 鏈核酸合成方法得到的生成物，再以瓊脂糖電 奴 ▼ / /1 照片。上圖為反應時間一小時，下圖為反膺日吝 一 結果。 -守間三小時的Row 2 ·· Reaction product of Example 1 ··· Reaction product of Example 3 Figure 14 As the target M13mpl8 concentration changes, ether 1 ♦ The product obtained by inventing the method for synthesizing single-strand nucleic acid, and then Agarose electric slave ▼ / / 1 photo. The upper picture shows the reaction time of one hour, and the lower picture shows the result of the next day. -Three hours

1 ：M13mpl8 dsDNA lx 1 0~15mol / tube 行2 :M13mpl8 dsDNA lx l〇-16mol/tube "ίτ3 ：M13mpl8 dsDNA lx 10~17mol/tube 行4 :M13mpl8 dsDNA lx 10_18mol/tube 行5 :M13mpl8 dsDNA lx l〇-19mol/tube 行6 :M13mpl8 dsDNA lx 10-2Gmol/tube 行7 :M13mpl8 dsDNA lx 10~21mol/tube 行8 :M13mpl8 dsDNA lx 10_22mol/tube 行9 :無標的物 行1 0 : X I V標準品標記 圖1 5為變異的位置及標的鹽基序列（標的物）相對各領 域之位置關係表示圖。下線表示鳥嘌呤（gUanine)在變異 型時以腺嘌呤（a d e n i n e )置換。 ' 圖1 6表示本發明增幅反應之生成物的瓊脂糖電泳結果 照片。 ° Μ ： 1〇〇 bp ladder (New England Biolabs) N :無模板（精製水） WT :野外株模板M13mpl8 1 fmol1: M13mpl8 dsDNA lx 1 0 ~ 15mol / tube line 2: M13mpl8 dsDNA lx l0-16mol / tube " ίτ3: M13mpl8 dsDNA lx 10 ~ 17mol / tube line 4: M13mpl8 dsDNA lx 10_18mol / tube line 5: M13mpl8 dsDNA lx l〇-19mol / tube row 6: M13mpl8 dsDNA lx 10-2Gmol / tube row 7: M13mpl8 dsDNA lx 10 ~ 21mol / tube row 8: M13mpl8 dsDNA lx 10_22mol / tube row 9: standardless row 10: XIV standard Labeling Figure 15 is a representation of the position of the mutation and the positional relationship between the target salt-based sequence (target) and various fields. The underline indicates that guanine (gUanine) is replaced with adenine (a d e n i n e) when it is mutated. 'Figure 16 shows a photograph of the results of agarose electrophoresis of the product of the amplification reaction of the present invention. ° Μ: 100 bp ladder (New England Biolabs) N: No template (purified water) WT: Field strain template M13mpl8 1 fmol

第48頁 1221855Page 48 1221855

MT 各 方 •變異株模板M13mpl8 1 fm〇i 圖1 7為標的mRNA相對應的鹽基序列中 鹽基序列的位置關係表示圖。 冓成I核苷酸 圖18表示將mRNA作為標# ’以 法得到的生成物，再以填脂糖電泳所得結酸合成 f施發明的最佳形態 免施例1 M13mp 1 8内之雙^的增幅 = M13mpl8作為模板，試驗本發明單股鏈上互補鹽某 $列父互連結的核酸合成方法。實驗中所使用的引子^為" M13FA ^M13RA ^M13F3^M13R3^4« 〇M13F3aM13R^J, 由作為合成起點的M13FA及M13RA得到的第一個核酸進行^ 換的外引子。外引子在較M13FA(或M13RA)後面必須作為^ 補鏈合成起點的引子，所以設計在M13FA(或M13RA)相鄰合 領域利用連續堆疊之現象。此外，為使M13FA(或mura)韻 先結合’引子的濃度設計得較高。 構成各引子的鹽基序列顯示於序列表。引子的構造本 徵歸納如下。且編碼標的（標的物）鹽基序列之各領域位3 關係圖表示於圖7。 引子 5側的領域/3’側的領域 M13FA與M13FA合成互補鏈之領域Fic相同 /與M13mpl8的領域F2C互補 M13RA與M13RA合成互補鏈之領域Ric相同Each side of MT • Mutant strain template M13mpl8 1 fm〇i Figure 17 shows the positional relationship of the base sequence in the base sequence corresponding to the target mRNA. Figure 18 shows the nucleotides. Figure 18 shows the product obtained by the method labeled with mRNA, and then synthesized by acid-binding acid electrophoresis. The best form of the present invention is exempt from Example 1 M13mp 1 8 Increase = M13mpl8 as a template to test the method for synthesizing nucleic acid of a parent interconnect of a column of complementary salts on a single strand of the present invention. The primers used in the experiments are " M13FA ^ M13RA ^ M13F3 ^ M13R3 ^ 4 «〇M13F3aM13R ^ J, the exon primers which were replaced by the first nucleic acid obtained from M13FA and M13RA as the starting point of synthesis. Outer primers must be behind the M13FA (or M13RA) primers that must be used as the starting point of ^ complement synthesis, so the design is to use the phenomenon of continuous stacking in the M13FA (or M13RA) adjoining domain. In addition, the concentration of the M13FA (or mura) rhyme-bound primer was designed to be high. The base sequence constituting each primer is shown in the sequence listing. The structural features of the primers are summarized as follows. And the relationship between bit 3 of each field encoding the base sequence of the target (target) is shown in FIG. 7. Primer 5 side domain / 3 ’side domain M13FA and M13FA synthesize complementary strands in the same domain Fic / M13mpl8 domain F2C complements the complementary domains M13RA and M13RA synthesize complementary strands the same Ric

/與M13FA合成互補鏈之領域ri c互補/ Complementary domain of ri c with M13FA

第49頁 1221855 五、發明說明（47)Page 49 1221855 V. Description of the invention (47)

Ml 3F3 與M13mpl8領域F2c之3’側鄰接的F3c互補 Μ1 3 R 3 與M13FA合成互補鍵領域F 2 c之3侧鄰接的r 3 c互 補 利用這樣的引子，由Ml 3mp 1 8之領域F 1 c開始至r】 上c的 範圍與其互補鹽基序列，以含有F2c之環狀構造形成序 列，進行單股鏈上交互連結的核酸合成。以此處之弓丨 行本發明核酸合成反應的反應液組成列示於下。 反應液組成（25 //L中） 20mM Tris-HC1 ρΗ8·8 lOraM KC1Ml 3F3 is complementary to the F3c adjacent to the 3 'side of the F2c in the M13mpl8 domain. M1 3 R 3 is complementary to the M13FA to synthesize the complementary bond domain F 2 c. The 3 adjacent r 3 c is complementary to each other. Using such a primer, the domain F 1 from Ml 3mp 1 8 c starts to r] The range of c above and its complementary base sequence forms a sequence with a cyclic structure containing F2c for nucleic acid synthesis of cross-linking on a single strand. The composition of the reaction solution that performs the nucleic acid synthesis reaction of the present invention with the bow here is shown below. Composition of reaction solution (25 / L) 20mM Tris-HC1 ρΗ8 · 8 lOraM KC1

lOmM (NH4)2S04 6mM MgS04 0.1% Tri ton X-l00 5% 二曱基亞碾（dimethylsulfoxide，DMSO) 0.4mM dNTPs 引子：lOmM (NH4) 2S04 6mM MgS04 0.1% Tri ton X-l00 5% dimethylsulfoxide (DMSO) 0.4mM dNTPs Primer:

800nM Ml 3FA/ 序列編號：1 800nM Ml 3RA/ 序列編號：2 200nM Ml 3F3/ 序列編號：3 20 0nM Ml 3R3/ 序列編號：4 標的：M13mpl8 dsDNA/序列編號：5 反應：上述反應液於95 °C加熱5分鐘，使標的變性成 為單股鏈形式。反應液移到冰上，添加4 U的B s t D N A聚人 酉母（New England Biolabs)，於65 °C反應1小時。反應後800nM Ml 3FA / Sequence number: 1 800nM Ml 3RA / Sequence number: 2 200nM Ml 3F3 / Sequence number: 3 20 0nM Ml 3R3 / Sequence number: 4 Target: M13mpl8 dsDNA / Sequence number: 5 Reaction: The above reaction solution is at 95 ° C was heated for 5 minutes to make the target denatured into a single strand. The reaction solution was moved to ice, and 4 U of B s t D N A was added to human diarrhea (New England Biolabs), and the reaction was performed at 65 ° C for 1 hour. After reaction

第50頁 1221855 五、發明說明（48) 於8 0 °C 1 0分鐘使反應停止，再將之移至冰上。 反應的確認：取上述反應液5 // L添加1 // L的裝載緩衝 液，採用2%瓊脂糖凝膠（0· 5%ΤΒΕ)，以1小時、80 mV進行 電泳。作為分子大小標定的是採用XIV (100 bp ladder， Boehringer Mannheim)。電泳後的膠體以SYBR Green I (Mo 1 ecu 1 ar Probes， I nc.)染色核酸而確認。結果顯示於 圖8。各行對應於以下的樣本。 1 · X I V標準品標記Page 50 1221855 V. Description of the invention (48) Stop the reaction at 80 ° C for 10 minutes, and then move it to ice. Confirmation of the reaction: Take 5 // L of the above reaction solution and add 1 // L of loading buffer, using 2% agarose gel (0.5% ΤΒΕ), and perform electrophoresis at 80 mV for 1 hour. For molecular size calibration, XIV (100 bp ladder, Boehringer Mannheim) was used. The colloid after electrophoresis was confirmed by staining the nucleic acid with SYBR Green I (Mo 1 ecu 1 ar Probes, Inc.). The results are shown in Figure 8. Each row corresponds to the following sample. 1X I V standard mark

2. 1 fmol M13mpl8 dsDNA 3. 無標的物 行3是確認未反應引子染色以外並沒有染色帶 (band)。行2是在標的存在的情形下，大小較小的染色帶 之梯狀排列（ladder )與大小較大的染色帶之污痕狀態 (smear)染色，及膠體内幾乎沒有泳動之生成物染色帶的 確認。大小較小的染色帶中，290bp、450bp附近的染色帶 分別是本發明合成反應的預測產物。序列編號·· 11及序列 編號：12是雙股鏈（相當於圖2-（ 7)及圖2-(10)的雙股鏈） 與序列編號·· 1 3 (相當於圖3 - ( 9 )中長的單股鏈）的大小是 一致的，故可以確認反應的進行與預測相符。大小較大的 污痕（smear)染色狀態（pattern)及不泳動的染色帶是因本 反應基本上是連續進行的反應，反應產物可能不是一定的 大小，且部分的單股鏈或是雙股鏈會形成複合體之類的複 雜構造’結果就形成這樣的電泳結果。 實施例2 反應產物的限制酶切割硿認2. 1 fmol M13mpl8 dsDNA 3. Non-standard substance Line 3 confirms that there is no band except for unreacted primer staining. Line 2 is the staining of the ladder-like arrangement of the smaller-sized stained bands and the smear staining of the larger-sized stained bands in the presence of the target, and the staining of the product with little swimming in the colloid. confirm. Among the smaller-sized staining bands, the staining bands around 290bp and 450bp are the predicted products of the synthesis reaction of the present invention, respectively. Sequence number 11 and sequence number 12 are double-stranded chains (equivalent to the double-stranded chains in Figures 2- (7) and Figure 2- (10)) and sequence numbers ·· 1 3 (equivalent to Figures 3-(9 The size of the medium-long single-strand chain) is consistent, so you can confirm that the progress of the reaction is consistent with the prediction. Larger smear dyeing patterns and non-swimable dyeing bands are basically continuous reactions. The reaction products may not be of a certain size, and part of the single or double strands Complex structures such as complexes will be formed as a result of such electrophoretic results. Example 2 Restriction enzyme cleavage of reaction product

第51頁 1221855 五、發明說明（49) 為達到了解實施例1得到之本發明單股鏈上互補鹽基 序列交互連結核酸構造的目的，進行限制酶消化。使用限 制酶的消化，理論上會一方面產生斷片，另一方面在實施 例1中所觀察到大小較大的污痕染色狀態及不泳動的染色 帶將消失，而由這些證據推定為本發明合成之單股鏈上互 補鹽基序列交互連結的核酸。 將八份實施例1的反應液（2 0 0 // L)收集在一起，經苯 酚（phenol)處理後，再進行酒精（ethanol)沈澱的精製。 回收其沈澱物以2 0 0 // L的TE緩衝液再溶解，取其中1 〇 # l 分別以BamHI、Pvul I及Hindi 11在37。(：消化2小時。消化物 採用2°/。的瓊脂糖（〇· 5%TBE)，經1小時，以80mV進行電泳。 作為分子大小標定的是採用SUPER LADDER-L0W( 10Obp ladder，Gensura laboratories， Inc·製）。電泳後的膠 體以SYBR Green I (Molecular Probes， Inc·)染色核酸 而確認。結果顯示於圖9。各行對應於以下的樣本。 1· SUPER LADDER-LOW 標準品標記 2. 精製物的BamHI消化物 3· 精製物的Pvul I消化物 4. 精製物的Hindi I I消化物 此處構成鏈長較為短之增幅生成物的鹽基序列推測是 序列編號：1 3、序列編號：14、序列編號：1 5及序列編 號：1 6等。由此處的鹽基序列推測各限制酶消化斷片的大 小列於表1。表中的L表示為含有環狀構造（單股鏈）的斷 片，故未確定其泳動位置。Page 51 1221855 V. Explanation of the invention (49) In order to understand the structure of the complementary salt sequence on the single strand of the present invention obtained in Example 1 to cross-link the nucleic acid structure, restriction enzyme digestion was performed. Digestion with restriction enzymes theoretically produces fragments on the one hand, and on the other hand, the stained state of larger stains and non-swimable staining bands observed in Example 1 will disappear, and it is presumed from this evidence that the synthesis of this invention Nucleic acid with complementary base sequences on a single strand. Eight reaction liquids (2 0 // // L) of Example 1 were collected together and treated with phenol, followed by refining by ethanol precipitation. The precipitate was recovered and re-dissolved in 2 0 // L of TE buffer, and 10 # l of them were taken at 37 with BamHI, Pvul I, and Hindi 11 respectively. (: Digestion for 2 hours. Digestion was performed at 2 ° / ° agarose (0.5% TBE), and electrophoresis was performed at 80 mV for 1 hour. The molecular size was calibrated using SUPER LADDER-L0W (10Obp ladder, Gensura laboratories , Inc.). The colloid after electrophoresis was confirmed by staining the nucleic acid with SYBR Green I (Molecular Probes, Inc.). The results are shown in Figure 9. Each line corresponds to the following sample. 1. SUPER LADDER-LOW standard label 2. BamHI digest of refined product 3. Pvul I digest of refined product 4. Hindi II digest of refined product Here the salt-based sequence constituting the amplification product with a shorter chain length is presumed to be a sequence number: 1 3, a sequence number: 14. Sequence number: 15 and sequence number: 16 etc. The size of each restriction enzyme digestion fragment estimated from the salt-based sequence here is listed in Table 1. L in the table indicates that it contains a circular structure (single strand). Segment, so its swimming position has not been determined.

第52頁 1221855 五、發明說明（50) 色丄本發明笮幅生成物的限制酶消化斷片 立列編號 BamHI Pvul Hi n dill 13 177+L 56+L 147+L 14 15+101+L - 142+L 15 171+101+L 56+L 147+161+L 16 11+101+230+L 237+L 142+170+L 义納結果 101, 177, 230 56, 237 142, 147, 161, 170 G1，15;無法確認) ^ 未消化的染色帶幾乎在消化後都與之前推定的大小有 隻化’所以確認目的反應產物有增幅。此外亦顯示幾乎沒 有非特異性產物。Page 52 1221855 V. Description of the invention (50) Color restriction enzyme digestion fragment of the present invention, column number BamHI Pvul Hi n dill 13 177 + L 56 + L 147 + L 14 15 + 101 + L-142 + L 15 171 + 101 + L 56 + L 147 + 161 + L 16 11 + 101 + 230 + L 237 + L 142 + 170 + L Inner results 101, 177, 230 56, 237 142, 147, 161, 170 G1, 15; cannot be confirmed) ^ The undigested stained bands are almost the same size as the previously estimated size after digestion, so it is confirmed that the target reaction product has increased. It also showed few non-specific products.

增幅反應液中添加甜菜碱（SIGMA)，進行對核酸增幅 反應效果的調查實驗。與實施例i相同，以M13mpl8作為模 板丄進行不同濃度甜菜碱下之本發明單股鏈上互補鹽基序 列父互連結的核酸合成方法。實驗中所用的引子與實施例 1使用的相同。模板DNA的量為10_21mol (M13mpl8)，而陰 性對照用水代替。添加的甜菜碱以〇、〇 · 5、1、2M的濃度 加入反應液’反應液組成列示於下。 反應液組成（2 5 /z L中）Betaine (SIGMA) was added to the amplification reaction solution to investigate the effect of nucleic acid amplification reaction. In the same manner as in Example i, a nucleic acid synthesis method using the parent salt of the complementary salt motif sequence on the single strand of the present invention under different concentrations of betaine was performed using M13mpl8 as a template. The primers used in the experiment were the same as those used in Example 1. The amount of template DNA was 10-21 mol (M13mpl8), and the negative control was replaced with water. The added betaine was added to the reaction solution at a concentration of 0, 0.5, 1, 2 M. The composition of the reaction solution is shown below. Composition of reaction solution (2 5 / z L)

第53頁 1221855 五、發明說明（51) 20mM Tris-HCl ρΗ8· 8 4mM MgS04 0·4mM dNTPs lOmM KC1 lOmM (NH4)2S04 0.1% Triton X-100 引子： 800nM M13FA/ 序列編號：1 800nM Ml 3RA/ 序列編號：2 200nM M13F3/ 序列編號：3 20 0nM M13R3/ 序列編號：4 標的：M13mpl8 dsDNA/序列編號：5 反應後電泳條件都與實施 使用之聚合酶、反應條件、 例1中所記載的相同。 結果顯示於圖1〇。甜菜5咸濃度〇·5、ι·⑽存在下的反 應有增幅產物量增大的效果。此外甜菜碱增加至2 〇Μ為 止，相反的，增幅產物並無法被確認。由此可見，適度的 甜菜碱存在下，結果顯示會有促進增幅反應的效果。甜菜 碱濃度2 Μ的情形下，考慮增幅產物低下的原因可能是τ m太 低所致。 實施例4 Η B V基因序列之增幅 以組合有HBV基因部份序列的M13mpl8作為模板，試驗 本發明的核酸合成方法。實驗所使用的引子為HB65FA(序 列編號：6)、HB65RA(序列編號·· 7) ' HBF3(序列編號：Page 53 1221855 V. Description of the invention (51) 20mM Tris-HCl pH 8. 8 4mM MgS04 0.4mM dNTPs 10mM KC1 10mM (NH4) 2S04 0.1% Triton X-100 Primer: 800nM M13FA / Sequence number: 1 800nM Ml 3RA / Sequence number: 2 200nM M13F3 / Sequence number: 3 20 0nM M13R3 / Sequence number: 4 Target: M13mpl8 dsDNA / Sequence number: 5 The electrophoresis conditions after the reaction are the same as those described in the polymerase, reaction conditions, and Example 1 used in the implementation . The results are shown in Figure 10. The response of beet 5 salty concentration 0.5, ι · ⑽ has the effect of increasing the amount of the product. In addition, the increase of betaine is not more than 2M. On the contrary, the increase product cannot be confirmed. It can be seen that, in the presence of moderate betaine, the results show that it has the effect of promoting the amplification reaction. In the case of betaine concentration of 2 M, the reason for considering the low amplification product may be that τ m is too low. Example 4 增 Amplification of B V gene sequence The M13mpl8 combined with the HBV gene partial sequence was used as a template to test the nucleic acid synthesis method of the present invention. The primers used in the experiments were HB65FA (sequence number: 6), HB65RA (sequence number · 7) 'HBF3 (sequence number:

第54頁 1221855 五、發明說明（52) 8)、HBR3(序列編號：9)等4種。HBF3與HBR3分別以HB65FA 及HB65RA作為合成起點而得到第一個核酸進行置換的外引 子。外引子在較HB65FA(或HB65RA)後面之處必須作為互補 鏈合成起點的引子，所以設計在HB65FA(或HB65RA)相鄰的 領域利用連續堆疊之現象。此外，為使HB65FA(或HB65RA) 優先結合，引子濃度設計得較高。在M13mpl8組合之HBV的 由來顯示於本實施例標的序列（4 3 0 b p )的序列1 0。 構成各引子的鹽基序列顯示於序列表。引子的構造特 徵歸納如下。且編碼標的鹽基序列之各領域位置關係圖表 不於圖1 1。 引子 5’侧的領域/3’側的領域 HB65FA 與HB6 5FA合成互補鏈之領域Flc相同 /與1^¥-诞1311^18的領域？2(3互補 HB6 5RA 與HB6 5RA合成互補鏈之領域Ric相同 /與HB65FA合成互補鏈之領域R2C互補 HBF3 與HBV-M13mpl8領域F2c之3,侧鄰接的F3c互補 HBR3 與HB65FA合成互補鏈領域R2c之3’側鄰接的R3c互 補 利用這樣的引子，由組合有HBV基因部份序列的 M13mpl8 (HBV-M13mpl8)之領域pic開始至Rlc的範圍與其 互補鹽基序列，以含有F2c之環狀構造形成序列進行單股 鏈上交互連結的核酸之合成。除了採用上述引子，其他都 是在與實施例1相同的條件下進行反應，此反應液再經瓊 脂糠電泳分析。結果顯示於圖丨2。各行對應於下列樣本。Page 54 1221855 V. Description of the invention (52) 8), HBR3 (serial number: 9), etc. 4 kinds. HBF3 and HBR3 use HB65FA and HB65RA as the starting points of synthesis, respectively, to obtain the first nucleic acid replacement primer. Outer primers must be used as the starting point of the complementary strand synthesis behind HB65FA (or HB65RA). Therefore, the design is to use the phenomenon of continuous stacking in areas adjacent to HB65FA (or HB65RA). In addition, for preferential binding of HB65FA (or HB65RA), the primer concentration is designed to be high. The origin of the HBV combined in M13mpl8 is shown in the sequence 10 of the target sequence (4 3 0 b p) in this example. The base sequence constituting each primer is shown in the sequence listing. The structural features of the primers are summarized as follows. Moreover, the positional relationship diagram of each field of the coding base sequence is not shown in FIG. 5 ′ side of the primer / 3/3 side of the primer HB65FA and HB6 5FA synthesize the complementary strand in the same Flc / 1 ^ ¥ -natal 1311 ^ 18? 2 (3 Complementary HB6 5RA and HB6 5RA synthesize complementary strands in the same Ric area / Synthetic complementary strands with HB65FA R2C complements HBF3 and HBV-M13mpl8 domains F2c 3, side adjacent F3c complements HBR3 and HB65FA synthetic complements R2c The 3 'side adjacent R3c is complementary using such a primer, starting from the field pic of M13mpl8 (HBV-M13mpl8) combined with the HBV gene partial sequence to the range of Rlc and its complementary base sequence, forming a sequence with a circular structure containing F2c The synthesis of nucleic acids that are linked on a single strand was performed. Except for the primers described above, the reaction was performed under the same conditions as in Example 1. This reaction solution was analyzed by agar bran electrophoresis. The results are shown in Figure 丨 2. Each row Corresponds to the following samples.

第55頁 1221855 五、發明說明（53) 1 · XIV標準品標記Page 55 1221855 V. Description of the invention (53) 1 · XIV standard mark

2. 1 fmol HBV-M13mpl8 dsDNA 3. 無標的物 與實施例1相同的，標的物存在的狀態下，可確認大 小較小的染色帶之梯狀排列（1 adder )與大小較大的染色帶 之污痕狀態染色，及膠體内幾乎沒有泳動之生成物染色帶 (行2)。大小較小的染色帶中，31 Obp、及480bp附近的染 色帶分別是本發明合成反應的預期產物。由於序列編號： 1 7及序列編號：1 8的雙股鏈大小一致，故可以確認反應的 進行與預測相符。大小較大的污痕染色狀態及不泳動的染 色帶，同於實施例1所述的結果，推測是因本發明特徵的 合成生成物構造而造成。依據此實驗可確認，即使增幅的 序列（標的物）不同，本發明的實施也是可行的。 實施例5 合成反應生成物大小（s i z e )之4認 為確δ忍基於本發明合成的核酸構造’在長期驗變性條 件下進行電泳分析。取實施例1及實施例4之標的存在下的 反應液5 // L，分別添加1 // L的鹼性裝載緩衝液，採用〇. 70/〇 瓊脂糖膠體（50 mM NaOH，ImM EDTA)，經14小時，以50 mA進行電泳。分子大小標定採用λ phage的Hindi I I消化斷 片。電泳後的膠體以1M Tris ρΗ8中和後，再用SYBR Green I (Molecular Probes，Inc·)染色核酸進行確認。 結果顯示於圖1 3。各行對應於以下各樣本。 1. 入Phage的Hindi 11消化斷片 2. 實施例1的反應生成物2. 1 fmol HBV-M13mpl8 dsDNA 3. The targetless substance is the same as in Example 1. In the state of the target substance, it is possible to confirm the ladder-like arrangement (1 adder) of the smaller-sized staining bands and the larger-sized staining bands. Stain staining, and little staining of the product in the colloid (line 2). Among the smaller-sized dyeing bands, the dyeing bands near 31 Obp and 480 bp are the expected products of the synthesis reaction of the present invention, respectively. Since the size of the double strands of the sequence number: 17 and the sequence number: 1 are the same, it can be confirmed that the progress of the reaction is consistent with the prediction. The stained state of larger stains and the non-swimable dyeing band are the same as the results described in Example 1, and are presumably caused by the structure of the synthetic product characteristic of the present invention. Based on this experiment, it can be confirmed that the implementation of the present invention is feasible even if the sequence of amplification (target) is different. Example 5 4 of the size (s i z e) of the synthesis reaction product was considered to be δ tolerance. The nucleic acid structure based on the synthesis of the present invention was subjected to electrophoretic analysis under long-term denaturation conditions. Take 5 // L of the reaction solution in the presence of the target of Example 1 and Example 4, and add 1 // L of alkaline loading buffer, using 0.70 / 0 agarose colloid (50 mM NaOH, ImM EDTA) After 14 hours, electrophoresis was performed at 50 mA. Molecular size calibration was performed using Hindi I I digestion fragments of λ phage. The gel after electrophoresis was neutralized with 1M Tris ρΗ8, and then the nucleic acid was confirmed by staining with SYBR Green I (Molecular Probes, Inc.). Results are shown in Figure 1 3. Each row corresponds to the following samples. 1. Hindi 11 digestion section with Phage 2. Reaction product of Example 1

第56頁 !221855Page 56! 221855

五、發明說明（54) ' 3·實施例4的反應生成物 反應生成物在鹼變性條件下電泳時，結果可以確認單 股鏈狀態的DNA大小。可以確認實施例丨（行2 )、實施例 4(行3)原本主生成物是在2kbase以内。此外，本發明生成 物在此分析法可確認領域内，可以確定至少延長反應可以 到6 kbase以上。再加上，實施例1及實施例4未變性條件下 不泳動之染色帶可以經變性狀態改變，分別分離為單股 鏈’大小也變得較小，而可確認為反應生成物。 免施例6 Ml 3mpl3内之領域的增幅中標的物澧廣依在之巧 幅的確認 進行本發明核酸之合成方法時，觀察標的物濃度變化 的影響。作為標的物的M13mpl8 dsDNA濃度以0-1 fm〇i， 反應時間以1小時及3小時進行，其他條件皆與實施例1相 同而實施本發明核酸合成方法。與實施例1相同的，在2 % 瓊脂糖膠體（0·5%ΤΒΕ)下進行電泳，再用SYBR Green I (Molecular Probes， Inc·)染色核酸進行確認。作為分子 大小標定的是採用XIV (100bp ladder, Boehringer Mannheim)。結果顯示於圖14 (上：反應時間1小時，下： 反應時間3小時）。各行對應於以下各樣本。 1. M13mpl8 dsDNA lx 1 0'15mo 1 /tube 2. M13mpl8 dsDNA lx 1 0'16mo 1 /tube 3. M13mpl8 dsDNA lx 1 0'17mo 1 /tube 4. M13mpl8 dsDNA lx 1 0'18mo 1 /tube 5. M13mpl8 dsDNA lx 1 0"19mo 1 /1ube5. Description of the invention (54) '3. The reaction product of Example 4 When the reaction product was electrophoresed under alkaline denaturing conditions, the size of the DNA in the single strand state was confirmed as a result. It can be confirmed that the original main product of Example 丨 (line 2) and Example 4 (line 3) is within 2kbase. In addition, the product of the present invention can confirm that the extension reaction can be at least 6 kbase in the field confirmed by this analysis method. In addition, the non-swimable dyeing bands in the undenatured conditions of Examples 1 and 4 can be changed into denatured states and separated into single strands, respectively. The size of the strands also becomes smaller, and they can be confirmed as reaction products. Exemption Example 6 Amplification in the field within Ml 3mpl3 The confirmation of the wide range of the winning target The confirmation of the wide range When performing the method for synthesizing the nucleic acid of the present invention, the effect of the concentration change of the target was observed. The target M13mpl8 dsDNA concentration was 0-1 fm0i, and the reaction time was performed at 1 hour and 3 hours. The other conditions were the same as in Example 1 and the nucleic acid synthesis method of the present invention was performed. As in Example 1, electrophoresis was performed on 2% agarose colloid (0.5% TBE), and the nucleic acid was confirmed by staining with SYBR Green I (Molecular Probes, Inc.). For molecular size calibration, XIV (100bp ladder, Boehringer Mannheim) was used. The results are shown in Fig. 14 (upper: reaction time 1 hour, lower: reaction time 3 hours). Each row corresponds to the following samples. 1. M13mpl8 dsDNA lx 1 0'15mo 1 / tube 2. M13mpl8 dsDNA lx 1 0'16mo 1 / tube 3. M13mpl8 dsDNA lx 1 0'17mo 1 / tube 4. M13mpl8 dsDNA lx 1 0'18mo 1 / tube 5. M13mpl8 dsDNA lx 1 0 " 19mo 1 / 1ube

第57頁 1221855 五、發明說明（55) 6. M13mpl8 dsDNA lx 10_20mol/tube 7. M13mpl8 dsDNA lx 1 0-21 mo 1 /tube 8. M13mpl8 dsDNA lx 1 0'22mo 1 /tube 9. 無標的物 1 0 · XI V標準品標記Page 57 1221855 V. Description of the invention (55) 6. M13mpl8 dsDNA lx 10_20mol / tube 7. M13mpl8 dsDNA lx 1 0-21 mo 1 / tube 8. M13mpl8 dsDNA lx 1 0'22mo 1 / tube 9. No object 1 0 XI V standard mark

電泳呈像的下面可見到各行有共通的染色帶，這是未 反應的引子被染色的結果。反應時間不改變，標的物不存 在時完全不能觀察到增幅產物。標的物存在下，可以得到 標的物濃度依存性的增幅產物染色情形。此外，反應時間 越長’可確認得到較低濃度的增幅產物。 直施例7 點突變（d 〇 i n t m u t a t i ο η)的檢出 (1) M13mpl8FM (變異型）的製作 作為標的物的DNA採用M13mpl8(野生型）及 M13mpl8FM(變異型）。變異型M13mpl8FM之製作為使用u PCR in vitro Mutagenesis Kit(寶酒造），導入 1 個鹽基 的置換。之後，以基因定序確認序列。F 1領域的序列顯示 於下。 野生型：CCGGGGATCCTCTAGAGTCG(序列編號：19) 變異型：CCGGGGATCCTCTAGAGTCA(序列編號：20)Below the electrophoretic image, a common staining band can be seen in each row, which is the result of unreacted primers being stained. The reaction time does not change, and no amplification products can be observed at all when the target is not present. In the presence of the target, dyeing of the amplified product of the concentration of the target can be obtained. In addition, the longer the reaction time ', it was confirmed that a lower concentration of the amplified product was obtained. Example 7 Detection of a point mutation (d o n t m u t a t i ο η) (1) Preparation of M13mpl8FM (variant) M13mpl8 (wild type) and M13mpl8FM (variant) were used as the target DNA. The mutant M13mpl8FM was prepared using u PCR in vitro Mutagenesis Kit (made by Takara Shuzo) and introduced a base substitution. Thereafter, the sequence was confirmed by genetic sequencing. The sequence of the F 1 field is shown below. Wild type: CCGGGGATCCTCTAGAGTCG (sequence number: 19) Variant: CCGGGGATCCTCTAGAGTCA (sequence number: 20)

(2) 引子的設計 使用之引子為FA引子之Flc，領域5’末端因野生型或 變異型而異的鹽基。變異的位置及編碼標的鹽基序列（標 的物）的各領域位置關係示於圖1 5。 (3) 增幅反應(2) Design of primers The primers used are the FLc of the FA primer, and the base at the 5 'end of the field differs depending on the wild type or the mutant type. The positions of the mutations and the positions of the various fields encoding the target base sequence (target) are shown in Figure 15. (3) Amplification response

第58頁 1221855 五、發明說明（56) 以M13mpl8(野生型）及M13mpl8FM(變異型）作為模板， 組成以下所示個別特異的引子，進行模板特異的增幅反應 而進行實驗。 野生型增幅用引子組：FAd4、RAd4、F3、R3 變異型增幅用引子組·· FAMd4、RAd4、F3、R3 各引子的鹽基序列依照以下所列序列。 FAd4 ·· CGACTCTAGAGGATCCCCGGTTTTTGTTGTGTGGAATTGTGAGCGGAT (序列編號：2 1)Page 58 1221855 V. Description of the invention (56) Using M13mpl8 (wild type) and M13mpl8FM (variant type) as templates, the individual specific primers shown below are composed, and template specific amplification reactions are performed for experiments. Primer set for wild type amplification: FAd4, RAd4, F3, R3 Primer set for mutant type amplification ... The base sequence of each primer of FAMd4, RAd4, F3, R3 follows the sequence listed below. FAd4 ·· CGACTCTAGAGGATCCCCGGTTTTTGTTGTGTGGAATTGTGAGCGGAT (Serial Number: 2 1)

FAMd4 ： TGACTCTAGAGGATCCCCGGTTTTTGTTGTGTGGAATTGTGAGCGGAT (序列編號：22) RAd4 ： CGTCGTGACTGGGAAAACCCTTTTTGTGCGGGCCTCTTCGCTATTAC (序列編號：23) F3 : ACTTTATGCTTCCGGCTCGTA(序列編號：24) R3 : GTTGGGAAGGGCGATCG (序列編號：25) (4) M13mpl8之點突變的檢出 反應液組成依照以下所列。FAMd4: TGACTCTAGAGGATCCCCGGTTTTTGTTGTGTGGAATTGTGAGCGGAT (Serial Number: 22) RAd4: CGTCGTGACTGGGAAAACCCTTTTTGTGCGGGCCTCTTCGCTATTAC (Sequence Number: 23) F3: ACTTTATGCTTCCGGCTCGTA (Sequence Number: 24) R3: GTTGGGA Listed below.

第59頁 1221855 五、發明說明（57)Page 59 1221855 V. Description of the invention (57)

D2W 3.72juL 終滾度 10X Thermo pol bufer(NEB) 2.5^L 20mm Tns-HCl pH8.8 2.5mM dNTP 4juL lOmMKCl lOmM (NH4)2S〇4 6mM MgS04 0.1% TntonX-100 400 /iM lOOmM MgS04 〇.5fiL 4M B etaine 6.25juL 1M M13FAd4 pnmer(10pmol/ju L)又 M13FAMd4 primer(lOpmol/U L) L 800nM M13RAd4 primer(10pmol/ U L) 2M1 800nM M13F3 primer(10pmol/U L) 0·5β L 200nM M13R3 primer(10pmol/U L) 0.5y L 200nM 全曼 22// LD2W 3.72juL Final Roll 10X Thermo pol bufer (NEB) 2.5 ^ L 20mm Tns-HCl pH8.8 2.5mM dNTP 4juL 10M KCl 10 mM (NH4) 2S〇4 6mM MgS04 0.1% TntonX-100 400 / iM 100M MgS04 〇.5fiL 4M B etaine 6.25juL 1M M13FAd4 pnmer (10 pmol/ju L) and M13FAMd4 primer (lOpmol / UL) L 800nM M13RAd4 primer (10 pmol/ UL) 2M1 800nM M13F3 primer (10 pmol/UL) 0 · 5β L 200nM M13R3 primer (10 pmol/10pmol/ UL) 0.5y L 200nM Allman 22 // L

上述反應液中添加1 fmol(2 //L)的M13mpl8或是 M13mpl8FM，於95 °C加熱5分鐘，使標的物變性為單股鏈。 反應液移至冰上，添加1 (8U)的Bst DNA聚合酶（NewAdd 1 fmol (2 // L) of M13mpl8 or M13mpl8FM to the above reaction solution, and heat at 95 ° C for 5 minutes to denature the target substance into single strands. The reaction solution was moved to ice, and 1 (8U) of Bst DNA polymerase (New

England Biolabs)，於 68 °C 或是 68.5 °C 反應1 小時。反應 後，於8 0 °C 1 0分鐘使反應停止，再將之移至冰上。England Biolabs) at 68 ° C or 68.5 ° C for 1 hour. After the reaction, the reaction was stopped at 80 ° C for 10 minutes, and then moved to ice.

如圖16所示，採用作為FA引子的野生型FAd4時，觀察 野生型模板存在下增幅的效果。另一方面，採用作為FA引 子的變異型FAMd4時，觀察野生型模板存在下增幅的效 果。 由以上的結果，顯示利用本發明增幅反應可以有效率As shown in Fig. 16, when wild-type FAd4 was used as a FA primer, the effect of amplification in the presence of a wild-type template was observed. On the other hand, when a mutant FAMd4 was used as a FA primer, the effect of the increase in the presence of a wild-type template was observed. From the above results, it is shown that the amplification reaction of the present invention can be efficient.

第60頁 1221855 五、發明說明（58) 的檢出點突變。 實施例8 以mRNA作為標的的增幅反應 以mRNA作為標的物核酸，試驗本發明的核酸合成方 法。作為標的物的mRNA為發現***特異抗原（Prostate specific antigen; PSA)之細胞的***癌細胞株LNCaP cell (ATCC No· CRL- 1 740 )，及非發現細胞的慢性骨髓性 白血病細胞株K562 cell (ATCC No· CCL-243)，以 1 : 1 06 - 1 0 0 : 1 06的比例混合，採用Qiagen公司（德國）的 RNeasy Mini Kit抽全RNA。實驗使用的引子為PSAFA、 PSARA、PSAF3及PSAR3等4種。PSAF3及PSAR3為分別作為合 成起點的PSAFA及PSARA得到第一個核酸置換的外引子。此 外，為使PSAFA (或PSARA)優先結合，其引子濃度設計得 較高。構成各引子的鹽基序列依照以下所列的序列。 引子： PSAFA ： TGTTCCTGATGCAGTGGGCAGCTTTAGTCTGCGGCGGTGTTCTG(序列 編號：2 6 ) PSARA : TGCTGGGTCGGCACAGCCTGAAGCTGACCTGAAATACCTGGCCTG(序列 編號：2 7 ) PSAF3 ·· TGCTTGTGGCCTCTCGTG(序列編號：28) PSAR3 ·· GGGTGTGGGAAGCTGTG(序列編號：29)Page 60 1221855 V. Description of the invention (58) The point mutation was detected. Example 8 Amplification reaction using mRNA as a target The nucleic acid synthesis method of the present invention was tested using mRNA as a target nucleic acid. The target mRNA is a prostate cancer cell line LNCaP cell (ATCC No. CRL-1 740) in which prostate-specific antigen (PSA) cells are found, and a non-discovered chronic myeloid leukemia cell line K562 cell ( ATCC No. CCL-243), mixed at a ratio of 1: 1 06-1 0 0: 1 06, and used Qiagen (Germany) RNeasy Mini Kit to extract the whole RNA. The experimental primers were PSAFA, PSARA, PSAF3 and PSAR3. PSAF3 and PSAR3 are the first primers for the first nucleic acid substitution of PSAFA and PSARA, respectively, which are the starting points of synthesis. In addition, in order to preferentially bind PSAFA (or PSARA), the primer concentration is designed to be high. The base sequence constituting each primer is in accordance with the sequence listed below. Primer: PSAFA: TGTTCCTGATGCAGTGGGCAGCTTTAGTCTGCGGCGGTGTTCTG (sequence number: 2 6) PSARA: TGCTGGGTCGGCACAGCCTGAAGCTGACCTGAAATACCTGGCCTG (sequence number: 2 7) PSAF3 ·· TGCTTGTGGCCTCTCTGTG (sequence number: 28) •

引子的構造特徵歸納於下。還有，對於標的物mRNA相 對應之DNA鹽基序列的各引子位置關係，及限制酶Sau3AIThe structural characteristics of the primer are summarized as follows. In addition, the positional relationship of each primer of the DNA base sequence corresponding to the target mRNA and the restriction enzyme Sau3AI

第61頁 1221855 五、發明說明（59) 的認識部位顯示於圖丨7。Page 61 1221855 V. Recognition site of invention description (59) is shown in Figure 丨 7.

引子 PSAFA PSARA PSAF3 PSAR3 補 5’側的領域/3’側的領域 與PSAFA合成互補鏈之領域pic相同 /與標的鹽基序列的領域F2c互補 與PSARA合成互補鍵之領域Ric相同 /與PSAFA合成互補鍵之領域R2c互補 與標的鹽基序列領域F2c之3’側鄰接的F3c互補 與PSAFA合成互補鏈領域R2c之3’側鄰接的R3c互 本發明核酸合成方法的反應液組成列示於下· 反應液組成（2 5 // L中） 20mM Tris-HCl pH8.8 4mM MgS04 0.4mM dNTPs lOmM KC1 lOmM (NH4)2S04 0.1% Triton X-100 0.8M betainePrimer PSAFA PSARA PSAF3 PSAR3 Complement 5 'side domain / 3' side domain is the same as the domain of the PSAFA synthetic complementary chain pic is the same as the domain of the target base sequence F2c is complementary to the domain of the PSARA synthetic complementary bond Ric is the same as the domain of the PSAFA synthetic complementary The R2c domain of the bond is complementary to the 3 'side adjacent to the F2c side of the target base sequence domain. The F3c complement is complementary to the R3c adjacent to the 3' side of the PSAFA synthesis complementary strand domain. The reaction solution composition of the nucleic acid synthesis method of the present invention is shown below. Reaction Liquid composition (2 5 // in L) 20mM Tris-HCl pH8.8 4mM MgS04 0.4mM dNTPs 10mM KC1 10mM (NH4) 2S04 0.1% Triton X-100 0.8M betaine

5mM DTT 1600nM PSAFA & PSARA 引子 200nM PSAF3 & PSAR3 引子 8U Bst DNA聚合酶5mM DTT 1600nM PSAFA & PSARA primer 200nM PSAF3 & PSAR3 primer 8U Bst DNA polymerase

100U ReverTra Ace (Τ0Υ0Β0,日本) 5 // g 全RNA ---100U ReverTra Ace (Τ0Υ0Β0, Japan) 5 // g full RNA ---

1221855 五 '發明說明（60) 全部成分均在冰上混合。本實驗中是以mRNA(單股鏈） 作為標的物，所以省略加熱變性成單股鏈的步驟。反應於 65 °C進行45分鐘，再於85 °C5分鐘停止反應。反應終了 後，取5 // L的反應液，用2%瓊脂糖進行電泳，再以SYBR Green I 檢出。 結果顯示於圖1 8。各行對應於以下各樣本。 行 Bst RT LNCaP細胞數/1 〇6個的K562 1 - + 0 2 - + 101221855 Five 'Invention note (60) All ingredients are mixed on ice. In this experiment, mRNA (single strand) is used as the target, so the step of denaturing to single strand by heating is omitted. The reaction was performed at 65 ° C for 45 minutes, and then stopped at 85 ° C for 5 minutes. After the reaction is completed, take 5 // L of the reaction solution, run it on 2% agarose, and then detect it with SYBR Green I. The results are shown in Figure 18. Each row corresponds to the following samples. Number of Bst RT LNCaP cells / 1 06 K562 1-+ 0 2-+ 10

3 + - 〇 4 + - 1〇 7 + + 1〇 8取行6的反應液1 //L經Sau3AI消化之產物 9取行7的反應液1 //L經Sau3AI消化之產物 10 標定分子大小的 100bp ladder(New England B i olabs)3 +-〇4 +-1〇7 + + 108 The reaction solution 1 of line 6 // L is digested by Sau3AI 9 The reaction solution of line 7 is 1 // L is digested by Sau3AI 10 The molecular size is calibrated 100bp ladder (New England B i olabs)

Bst DNA聚合酶、ReverTra Ace中任何一項缺乏時， 增幅產物就無法得到（行1 - 4)。兩種酵素都存在下，由 LNCaP來的RNA也存在時，可檢出增幅產物（行5_了）。在 萬個K 5 6 2細胞中的1個L N C a P細胞之R N A抽出後仍可被檢出 (行6)。增幅產物以對標的物内部序列有限制酶切位的 Sau3AI消化，而得到預期大小的消化斷片（行8， 9)。In the absence of either Bst DNA polymerase or ReverTra Ace, amplification products are not available (lines 1-4). In the presence of both enzymes, amplification products can be detected when RNA from LNCaP is also present (line 5_). The R N A of 1 L N C a P cell out of 10,000 K 5 62 cells could still be detected after extraction (line 6). The amplified product was digested with Sau3AI, which has restriction sites for the internal sequence of the target, and digested fragments of the expected size were obtained (lines 8, 9).

第63頁 似 1855Page 63 Like 1855

田以上結果得知Takami results learned

作i^ ^ τπ似敗贫珉方法中，以KM =的物的情形下’確認仍然可以得到目的反應產物。 處利用的可能作 不兩本發明新式寡核苷酸的核酸合成方法時，提供了 ϊ:ί:ί度調控的單股鏈上交互互補鹽基序列連結的核 補二A 2。基於本發明的寡核苷酸作為引子所合成的互 互補铋I:以其本身作為模板，再以3,末端作為合成新的 i=;起點。此時，#到新引子之結合而伴隨形成 的太基3^、 *此。卩分開始的互補鏈合成，會先以合成而得 可妒点i ί模板，進行互補鏈合成，其生成物再經置換而 人對結合的可能狀態。這樣得到以本身做模板的 ，是如sda之類已知的核酸合成方法的組合，而 對核，合成效率有提高的貢獻。 知桉=f丄本發明之理想狀態下，不只單純的達成提高已 可期二的效率，也不需要複雜的溫度調控，而且 板法。也就是說，基於本發明寡核苷酸的模 交互ΐ:於與其對應的互補冑，且單股冑上互補鹽基序列 連、=核冑’可連續進行合成反應。這樣的反應，原 間由产Li ΐ必要原料用盡之前’可繼續不斷的合成，其 此造部分開始新核酸生成的步驟也繼續進行。如 為In，由％狀構造結合的寡核苷酸開始進行延長反應， ===也r多數組互補鹽基序列連結者)延長 丁 k仏3 -〇H的鏈置換反應。一方面，長單股鏈在In the i ^^ τπ-like failure method, it is confirmed that the target reaction product can still be obtained in the case of KM =. It may be used as a method for nucleic acid synthesis of the novel oligonucleotides of the present invention, and provides a core complement A 2 that is linked to a complementary complementary base sequence on a single strand regulated by a single strand. Mutual complementary bismuth I synthesized based on the oligonucleotide of the present invention as a primer: using itself as a template, and then using 3, the end as a new i =; starting point. At this time, the combination of # to the new primer is accompanied by the formation of Taiji 3 ^, * this. The synthesis of the complementary strand starting from the division will first be synthesized to obtain the enviable point i ί template, and the complementary strand synthesis will be performed, and the product thereof will be replaced and the human pair may be combined. In this way, the template obtained by itself is a combination of known nucleic acid synthesis methods such as sda, and it contributes to the improvement of nuclear and synthesis efficiency. Under the ideal state of the present invention, not only can the efficiency of the second phase be improved, but also the complicated temperature control is not required, and the plate method is not required. That is, based on the mode interaction of the oligonucleotides of the present invention: the complementary bases corresponding to them, and the complementary base sequence on a single strand of bases can be continuously synthesized. Such a reaction can continue to be synthesized until the necessary raw materials for Li 产 production are exhausted, and the steps for starting new nucleic acid generation in this part also continue. In the case of In, the elongation reaction is started by the% -bound structured oligonucleotide, and === also the r-array complementary salt sequence linker) elongates the chain displacement reaction of 仏 k 仏 3-OH. On the one hand, long single-strand chains

第64頁 1221855Page 64 1221855

〜，十、河 鏈的置換反 同時，而在 在2個連續 。這對特異 的設計與位 而開始進行 之特異性是 的敏感度精~, Ten, the replacement of the river chain at the same time, and in 2 consecutive. The specificity of this specific design and position begins with the sensitivity of

ι Η以本身作為模板而進行互補鏈合成反應 ^ j同時，由環狀構造開始進行新合成互補 =二廷樣的增幅反應工程，是維持高特異性的 寺溫條件下進行的。 、本發明的寡核苷酸是首次設計成可以配置 項域中而為本發明核酸合成反應之引子的機能 性的維持具有極大貢獻。例如PCR中，2個引子 置之間疋沒有關係的，比起非特異的錯誤配對 非2異增幅反應來說，本發明可期待具有較高 較容易說明的。利用這項特徵以SNPs可以較高 確檢出結果。 —本發明的特徵是使這樣的反應可經較單純的試藥構成 $容易達成。例如本發明的寡核苷酸雖說具有特殊的構 =二但鹽基序列也有選擇上的問冑，而物質上單純的以寡 乂甘酸為基礎。此外，理想中的狀態是可以採用鏈置換型 互補鏈合成反應之觸媒的DNA聚合酶來進行反應。還有、，ι 进行 Uses itself as a template to perform a complementary strand synthesis reaction ^ j At the same time, a new synthetic complementary = two-tingle-like amplification reaction project is started from a cyclic structure, and it is performed under high temperature conditions. 2. The oligonucleotide of the present invention is designed for the first time to be able to arrange the domain and maintain the function of the primer of the nucleic acid synthesis reaction of the present invention. For example, in PCR, there is no relationship between the two primer sets, and the present invention can be expected to have a higher and easier to explain than a non-specific mismatched non-2 heterogeneous amplification response. With this feature, SNPs can be used to confirm the results. -The present invention is characterized in that such a reaction can be easily achieved by a simpler reagent configuration. For example, although the oligonucleotide of the present invention has a special configuration, the salt-based sequence is also selective, and the material is purely based on oligoglyceric acid. In addition, ideally, a DNA polymerase that is a catalyst for a strand displacement type complementary strand synthesis reaction can be used for the reaction. and also,,

= 作為模板而實施本發明的情形時，利用以&1^人聚合 名之類同時具有反轉錄酶活性及鏈置換型DNA聚合酶活性 的ί素，可以讓全部的酵素反應都以單一酵素進行。這樣 以簡單的酵素反應實現高度核酸增幅反應的反應原理是前 所未知的。或者是，適用SDA等已知核酸合成反應，再與 士發=相組合，而不需新酵素，單純的組合基於本發明的 秦核苷酸，也可以適應各種反應系統。依據此說，本發明 的核酸合成方法在花費上較經濟亦為其優點。= In the case of implementing the present invention as a template, all enzyme reactions can be performed with a single enzyme by using a gene with both reverse transcriptase activity and strand displacement DNA polymerase activity, such as & 1 ^ human polymerization name. get on. In this way, the reaction principle for achieving a high degree of nucleic acid amplification reaction with a simple enzyme reaction is unknown. Alternatively, a known nucleic acid synthesis reaction such as SDA can be applied and combined with Shifa = without the need for a new enzyme. The simple combination can be adapted to various reaction systems based on the Qin nucleotide of the present invention. Based on this, the nucleic acid synthesis method of the present invention is also economical in terms of cost and has its advantages.

第65頁 1221855 五、發明說明（63) 由以上所述，本發明的核酸合成方法及其寡核苷酸可 說是提供了操作性容易（不需溫度調控）、合成效率提高、 經濟性、且高特異性，能同時解決多種困難的課題的新式 原理。Page 65 1221855 V. Explanation of the invention (63) From the above, the method for synthesizing nucleic acid and its oligonucleotide according to the present invention can be said to provide easy operability (no need for temperature control), improved synthesis efficiency, economical, The new principle is highly specific and can solve many difficult problems at the same time.

第66頁 1221855 1/12 序列表 <110> Eiken Kagaku Kabushiki Kaisha <120> Method for Synthesizing The Nucleic Acid.Page 66 1221855 1/12 Sequence Listing < 110 > Eiken Kagaku Kabushiki Kaisha < 120 > Method for Synthesizing The Nucleic Acid.

<130> E2-001PCT <140> <141> <150> JP-1998-317476 <151〉 1998-11-09< 130 > E2-001PCT < 140 > < 141 > < 150 > JP-1998-317476 < 151> 1998-11-09

<160〉 29 <170> Patentln Ver. 2.0 <210〉 1 <211〉 52 <212> DNA <213> Artificial Sequence $ <220> <223> Description of Artificial Sequence:Artificially synthesized primer sequense <400〉 1 cgactctaga ggatccccgg gtactttttg ttgtgtggaa ttgtgagcgg at< 160〉 29 < 170 > Patentln Ver. 2.0 < 210〉 1 < 211〉 52 < 212 > DNA < 213 > Artificial Sequence $ < 220 > < 223 > Description of Artificial Sequence: Artificially synthesized primer sequense < 400〉 1 cgactctaga ggatccccgg gtactttttg ttgtgtggaa ttgtgagcgg at

<210〉 2 <211> 51 <212> DNA <213> Artificial Sequence <220> 1221855 2/12 synthesized primer sequense <400> 2 acaacgtcgt gactgggaaa accctttttg tgcgggcctc ttcgctatta c< 210> 2 < 211 > 51 < 212 > DNA < 213 > Artificial Sequence < 220 > 1221855 2/12 synthesized primer sequense < 400 > 2 acaacgtcgt gactgggaaa accctttttg tgcgggcctc ttcgctatta c

<210〉 3 <211〉 21 <212〉 DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:Artificially synthesized primer sequense <400> 3 actttatgct tccggctcgt a< 210〉 3 < 211〉 21 < 212〉 DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Artificially synthesized primer sequense < 400 > 3 actttatgct tccggctcgt a

<210> 4 <211〉 17 <212> DNA <213> Artificial Sequence <220> · <223> Description of Artificial Sequence:Artificially synthesized primer sequense <400> 4 gttgggaagg gcgatcg <210> 5 -< 210 > 4 < 211> 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Artificially synthesized primer sequense < 400 > 4 gttgggaagg gcgatcg < 210 > 5-

<211〉 600 <212> DNA <213> Bacteriophage M13mpl8 <400> 5 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120 1221855 3/12 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg aattcgagct 240 cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagcttggca ctggccgtcg 300 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 360 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 420 agttgcgcag cctgaatggc gaatggcgct itgcctggtt tccggcacca gaagcggtgc 480 cggaaagctg gctggagtgc gatcttcctg aggccgatac ggtcgtcgtc ccctcaaact 540 ggcagatgca cggttacgat gcgcccatct acaccaacgt aacctatccc attacggtca 600 <210〉 6 <211> 63 i≪ 211> 600 < 212 > DNA < 213 > Bacteriophage M13mpl8 < 400 > 5 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120 1221855 3/12 cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180 tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg aattcgagct 240 cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagcttggca ctggccgtcg 300 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 360 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 420 agttgcgcag cctgaatggc gaatggcgct itgcctggtt tccggcacca gaagcggtgc 480 cggaaagctg gctggagtgc gatcttcctg aggccgatac ggtcgtcgtc ccctcaaact 540 ggcagatgca cggttacgat gcgcccatct acaccaacgt aacctatccc attacggtca 600 < 210> 6 < 211 > 63 i

<212> DNA <213> Artificial Sequence <220〉 <223> Description of Artificial Sequence:Artificially synthesized primer sequence <400> 6 ctcttccaaa agtaaggcag gaaatgtgaa accagatcgt aatttggaag acccagcatc 60 cag 63 <210> 7 · <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:Artificially synthesized primer sequence <400> 7 gtggattcgc actcctcccg ctgatcggga cctgcctcgt cgt 43< 212 > DNA < 213 > Artificial Sequence < 220> < 223 > Description of Artificial Sequence: Artificially synthesized primer sequence < 400 > 6 ctcttccaaa agtaaggcag gaaatgtgaa accagatcgt aatttggaag acccagcatc 60 cag 63 < 210 > 7 211 > 43 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Artificially generated primer sequence < 400 > 7 gtggattcgc actcctcccg ctgatcggga cctgcctcgt cgt 43

<210〉 8 <211> 16 <212〉 DNA 1221855 4/12 <213> Artificial Sequence <220> <223> Description of Artificial Sequence .-Artificially synthesized primer sequence <400> 8 gccacctggg tgggaa 16< 210〉 8 < 211 > 16 < 212〉 DNA 1221855 4/12 < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence .-Artificially synthesized primer sequence < 400 > 8 gccacctggg tgggaa 16

<210〉 9 <211> 22 <212〉 DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:Artificially synthesized primer sequence <400> 9 ggcgagggag ttcttcttct ag 22 <210> 10 <211〉 430 .< 210> 9 < 211 > 22 < 212> DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Artificially synthesized primer sequence < 400 > 9 ggcgagggag ttcttcttct ag 22 < 210 > 10 < 211〉 430.

<212〉 DNA <213〉 Hepatitis B virus · <400> 10 ctccttgaca ccgcctctgc tctgtatcgg cctcaccata cagcactcag gcaagctatt acctgggtgg gaagtaattt ggaagaccca aatgttaata tgggcctaaa aatcagacaa tttggaagag aaactgtttt ggagtatttg cccgcttaca gaccaccaaa tgcccctatc agacgacgag gcaggtcccc tagaagaaga tcgccgcgtc gaggccttag agtctccgga acattgttca 60 ctgtgttggg gtgagttaat gaatctggcc 120 gcatccaggg aattagtagt cagctatgtc 180 ctattgtggt ttcacatttc ctgccttact 240 gtatcttttg gagtgtggat tcgcactcct 300 ttatcaacac ttccggaaac tactgttgtt 360 actccctcgc ctcgcagacg aaggtctcaa 420 430 <210〉 11 1221855 5/12≪ 212> DNA < 213> Hepatitis B virus · < 400 > 10 ctccttgaca ccgcctctgc tctgtatcgg cctcaccata cagcactcag gcaagctatt acctgggtgg gaagtaattt ggaagaccca aatgttaata tgggcctaaa aatcagacaa tttggaagag aaactgtttt ggagtatttg cccgcttaca gaccaccaaa tgcccctatc agacgacgag gcaggtcccc tagaagaaga tcgccgcgtc gaggccttag agtctccgga acattgttca 60 ctgtgttggg gtgagttaat gaatctggcc 120 gcatccaggg aattagtagt cagctatgtc 180 ctattgtggt ttcacatttc ctgccttact 240 gtatcttttg gagtgtggat tcgcactcct 300 ttatcaacac ttccggaaac tactgttgtt 360 actccctcgc ctcgcagacg aaggtctcaa 420 430 < 210 21 12 12

<211> 293 <212〉 DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:Artificially synthesized sequence <400〉 11 acaacgtcgt gactgggaaa accctttttg tgcgggcctc ttcgctatta cgccagctgg 60 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac 120 gacgttgtaa aacgacggcc agtgccaagc ttgcatgcct gcaggtcgac tctagaggat 180 ccccgggtac cgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 240 tatccgctca caattccaca caacaaaaag tacccgggga tcctctagag teg 293< 211 > 293 < 212〉 DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Artificially synthesized sequence < 400> 11 acaacgtcgt gactgggaaa accctttttg tgcgggcccatc tggtagggggagggagg 120 gacgttgtaa aacgacggcc agtgccaagc ttgcatgcct gcaggtcgac tctagaggat 180 ccccgggtac cgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 240 tatccgctca caattccaca caacaaagag tacc

<210> 12 <211〉 293 <212> DNA <213> Artificial Sequence <220〉 <223> Description of Artificial Sequence:Artificially synthesized sequence <400> 12 · cgactctaga ggatccccgg gtactttttg ttgtgtggaa ttgtgagcgg ataacaattt 60 cacacaggaa acagctatga ccatgattac gaattegage tcggtacccg gggatcctct 120 agagtcgacc tgcaggcatg caagcttggc actggccgtc gttttacaac gtcgtgactg 180 ggaaaaccct ggcgttaccc aaettaateg ccttgcagca catccccctt tcgccagctg 240 gegtaatage gaagaggccc gcacaaaaag ggttttccca gtcacgacgt tgt 293< 210 > 12 < 211> 293 < 212 > DNA < 213 > Artificial Sequence < 220> < 223 > Description of Artificial Sequence: Artificially synthesized sequence < 400 > 12 · cgactctaga ggatccccgg gtactttttg ttgtgtggaa ttgtgagctt ggata cacacaggaa acagctatga ccatgattac gaattegage tcggtacccg gggatcctct 120 agagtcgacc tgcaggcatg caagcttggc actggccgtc gttttacaac gtcgtgactg 180 ggaaaaccct gg tgcc tgcc cctgcggcgca tcc

<210> 13 <211> 459 <212〉 DNA <213> Artificial Sequence <220〉 1221855 6/12 <223> Description of Artificial Sequence:Artificially synthesized sequence <400> 13 acaacgtcgt gactgggaaa accctttttg tgcgggcctc ttcgctatta cgccagctgg 60 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac 120 gacgttgtaa aacgacggcc agtgccaagc ttgcatgcct gcaggtcgac tctagaggat 180 ccccgggtac cgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 240 tatccgctca caattccaca caacaaaaag tacccgggga tcctctagag tcgacctgca 300 ggcatgcaag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 360 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 420 aggcccgcac aaaaagggtt ttcccagtca cgacgttgt 459 <210> 14 ·< 210 > 13 < 211 > 459 < 212〉 DNA < 213 > Artificial Sequence < 220〉 1221855 6/12 < 223 > Description of Artificial Sequence: Artificially synthesized sequence < 400 > 13 acaacgtcgt gactgggaaa accctttttg tgcgggcctc ttcgctatta cgccagctgg 60 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac 120 gacgttgtaa aacgacggcc agtgccaagc ttgcatgcct gcaggtcgac tctagaggat 180 ccccgggtac cgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 240 tatccgctca caattccaca caacaaaaag tacccgggga tcctctagag tcgacctgca 300 ggcatgcaag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 360 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 420 aggcccgcac aaaaagggtt ttcccagtca cgacgttgt 459 < 210 > 14 ·

<211> 458 <212〉 DNA <213> Artificial Sequence <220〉 <223> Description of Artificial Sequence .-Artificially synthesized sequence <400> 14 f cgactctaga ggatccccgg gtactttttg ttgtgtggaa ttgtgagcgg ataacaattt 60 cacacaggaa acagctatga ccatgattac gaattcgagc tcggtacccg gggatcctct 120 agagtcgacc tgcaggcatg caagcttggc actggccgtc gttttacaac gtcgtgactg 180 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 240 gcgtaatagc gaagaggccc gcacaaaaag ggttttccca gtcacgacgt tgtaaaacga 300 cggccagtgc caagcttgca tgcctgcagg tcgactctag aggatccccg ggtaccgagc 360 tcgaattcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 420 ccacacaaca aaaagtaccc ggggatcctc tagagtcg 458< 211 > 458 < 212〉 DNA < 213 > Artificial Sequence < 220〉 < 223 > Description of Artificial Sequence .-Artificially synthesized sequence < 400 > 14 f cgactctaga ggatccccgg gtactttttg ttgtgtgggga ttgtgagcgg ataacaattcggatcagatcagat tcggtacccg gggatcctct 120 agagtcgacc tgcaggcatg caagcttggc actggccgtc gttttacaac gtcgtgactg 180 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 240 gcgtaatagc gaagaggccc gcacaaaaag ggttttccca gtcacgacgt tgtaaaacga 300 cggccagtgc caagcttgca tgcctgcagg tcgactctag aggatccccg ggtaccgagc 360 tcgaattcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 420 ccacacaaca aaaagtaccc ggggatcctc tagagtcg 458

<210〉 15 <211> 790 <212〉 DNA <213> Artificial Sequence 1221855 7/12 <220〉 . <223> Description of Artificial Sequence .-Artificially synthesized sequence <400> 15 acaacgtcgt gactgggaaa accctttttg tgcgggcctc ttcgctatta cgccagctgg 60 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac 120 gacgttgtaa aacgacggcc agtgccaagc ttgcatgcct gcaggtcgac tctagaggat 180 ccccgggtac cgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 240 tatccgctca caattccaca caacaaaaag tacccgggga tcctctagag tcgacctgca 300 ggcatgcaag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 360 鬌 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 420 aggcccgcac aaaaagggtt ttcccagtca cgacgttgta aaacgacggc cagtgccaag 480 cttgcatgcc tgcaggtcga ctctagagga tccccgggta ctttttgttg tgtggaattg 540 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgaa ttcgagctcg 600 gtacccgggg atcctctaga gtcgacctgc aggcatgcaa gcttggcact ggccgtcgtt 660 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 720 ccccctttcg ccagctggcg taatagcgaa gaggcccgca caaaaagggt tttcccagtc 780 acgacgttgt 790 <210> 16 <211> 789 <212〉 DNA · <213> Artificial Sequence · <220〉 <223> Description of Artificial Sequence:Artificially synthesized sequence <400> 16 - cgactctaga ggatccccgg gtactttttg ttgtgtggaa ttgtgagcgg ataacaattt 60 cacacaggaa acagctatga ccatgattac gaattcgagc tcggtacccg gggatcctct 120 · agagtcgacc tgcaggcatg caagcttggc actggccgtc gttttacaac gtcgtgactg 180 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 240 · gcgtaatagc gaagaggccc gcacaaaaag ggttttccca gtcacgacgt tgtaaaacga 300 cggccagtgc caagcttgca tgcctgcagg tcgactctag aggatccccg ggtaccgagc 360 tcgaattcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 420 1221855 8/12 ccacacaaca aaaagtaccc ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg 480 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tttttgtgcg ggcctcttcg 540 ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca 600 gggttttccc agtcacgacg ttgtaaaacg acggccagtg ccaagcttgc atgcctgcag 660 gtcgactcta gaggatcccc gggtaccgag ctcgaattcg taatcatggt catagctgtt 720 tcctgtgtga aattgttatc cgctcacaat tccacacaac aaaaagtacc cggggatcct 780 ctagagtcg 789< 210〉 15 < 211 > 790 < 212〉 DNA < 213 > Artificial Sequence 1221855 7/12 < 220〉. < 223 > Description of Artificial Sequence .-Artificially synthesized sequence < 400 > 15 acaacgtcgt gactgggaaa accctttttg tgcgggcctc ttcgctatta cgccagctgg 60 cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac 120 gacgttgtaa aacgacggcc agtgccaagc ttgcatgcct gcaggtcgac tctagaggat 180 ccccgggtac cgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 240 tatccgctca caattccaca caacaaaaag tacccgggga tcctctagag tcgacctgca 300 ggcatgcaag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 360 Duo ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 420 aggcccgcac aaaaagggtt ttcccagtca cgacgttgta aaacgacggc cagtgccaag 480 cttgcatgcc tgcaggtcga ctctagagga tccccgggta ctttttgttg tgtggaattg 540 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgaa ttcgagctcg 600 gtacccgggg atcctctaga gtcgacctgc aggcatgcaa gcttggcact ggccgtcgtt 660 ttacaacgtc gtgactggga aaac cctggc gttacccaac ttaatcgcct tgcagcacat 720 ccccctttcg ccagctggcg taatagcgaa gaggcccgca caaaaagggt tttcccagtc 780 acgacgttgt 790 of < 210 > 210 > 212 > 212 > 212 artificially synthesized sequence < 400 > 16 - cgactctaga ggatccccgg gtactttttg ttgtgtggaa ttgtgagcgg ataacaattt 60 cacacaggaa acagctatga ccatgattac gaattcgagc tcggtacccg gggatcctct 120 · agagtcgacc tgcaggcatg caagcttggc actggccgtc gttttacaac gtcgtgactg 180 ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 240 · gcgtaatagc gaagaggccc gcacaaaaag ggttttccca gtcacgacgt tgtaaaacga 300 cggccagtgc caagcttgca tgcctgcagg tcgactctag aggatccccg ggtaccgagc 360 tcgaattcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 420 1221855 8/12 ccacacaaca aaaagtaccc ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg 480 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tttttgtgcg ggcctcttcg 540 ctattacgcc agctggcgaa agggggat gt gctgcaaggc gattaagttg ggtaacgcca 600 gggttttccc agtcacgacg ttgtaaaacg acggccagtg ccaagcttgc atgcctgcag 660 gtcgactcta gaggatcc cctcatggtgtggtgatctgat

<210> 17 <211> 310 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:Artificially synthesized sequence <400> 17< 210 > 17 < 211 > 310 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Artificially synthesized sequence < 400 > 17

gtggattcgc actcctcccg ctgatcggga cctgcctcgt cgtctaacaa cagtagtttc 60 cggaagtgtt gataagatag gggcatttgg tggtctgtaa gcgggaggag tgcgaatcca 120 cactccaaaa gataccaaat actccaaaac agtttctctt ccaaaagtaa ggcaggaaat 180 gtgaaaccac aatagttgtc tgatttttag gcccatatta acattgacat agctgactac 240 taattccctg gatgctgggt cttccaaatt acgatctggt ttcacatttc ctgccttact 300 tttggaagag 310gtggattcgc actcctcccg ctgatcggga cctgcctcgt cgtctaacaa cagtagtttc 60 cggaagtgtt gataagatag gggcatttgg tggtctgtaa gcgggaggag tgcgaatcca 120 cactccaaaa gataccaaat actccaaaac agtttctctt ccaaaagtaa ggcaggaaat 180 gtgaaaccac aatagttgtc tgatttttag gcccatatta acattgacat agctgactac 240 taattccctg gatgctgggt cttccaaatt acgatctggt ttcacatttc ctgccttact 300 tttggaagag 310

<210> 18 <211> 465 <212〉 DNA <213> Artificial Sequence <220〉 <223> Description of Artificial Sequence-.Artificially synthesized sequence <400> 18 gtggattcgc actcctcccg ctgatcggga cctgcctcgt cgtctaacaa cagtagtttc 60 cggaagtgtt gataagatag gggcatttgg tggtctgtaa gcgggaggag tgcgaatcca 120 1221855 9/12 cactccaaaa gataccaaat actccaaaac agtttctctt ccaaaagtaa ggcaggaaat 180 gtgaaaccac aatagttgtc tgatttttag gcccatatta acattgacat agctgactac 240 taattccctg gatgctgggt cttccaaatt acgatctggt ttcacatttc ctgccttact 300 tttggaagag aaactgtttt ggagtatttg gtatcttttg gagtgtggat tcgcactcct 360 cccgcttaca gaccaccaaa tgcccctatc ttatcaacac ttccggaaac tactgttgtt 420 agacgacgag gcaggtcccg atcagcggga ggagtgcgaa tccac 465 <210> 19 <211〉 20 <212> DNA <213> M13mpl8 <400> 19 ccggggatcc tctagagtcg 20< 210 > 18 < 211 > 465 < 212> DNA < 213 > Artificial Sequence < 220> < 223 > Description of Artificial Sequence-.Artificially synthesized sequence < 400 > 18 gtggattcgc actcctcccg ctgatcggga cctgcctcgt cgtctaca cggaagtgtt gataagatag gggcatttgg tggtctgtaa gcgggaggag tgcgaatcca 120 1221855 9/12 cactccaaaa gataccaaat actccaaaac agtttctctt ccaaaagtaa ggcaggaaat 180 gtgaaaccac aatagttgtc tgatttttag gcccatatta acattgacat agctgactac 240 taattccctg gatgctgggt cttccaaatt acgatctggt ttcacatttc ctgccttact 300 tttggaagag aaactgtttt ggagtatttg gtatcttttg gagtgtggat tcgcactcct 360 cccgcttaca gaccaccaaa tgcccctatc ttatcaacac ttccggaaac tactgttgtt 420 agacgacgag gcaggtcccg atcagcggga ggagtgcgaa tccac 465 < 210 > 19 < 211〉 20 < 212 > DNA < 213 > M13mpl8 < 400 > 19 ccggggatcc tctagagtcg 20

<210> 20 <211〉 20 <212> DNA <213> M13mpl8 mutant <400> 20 ccggggatcc tctagagtca 20 / <210> 21 <211> 48 ·< 210 > 20 < 211> 20 < 212 > DNA < 213 > M13mpl8 mutant < 400 > 20 ccggggatcc tctagagtca 20 / < 210 > 21 < 211 > 48 ·

<212〉 DNA <213> Artificial Sequence <220〉 <223> Description of Artificial Sequence:Artificially synthesized primer sequence <400> 21 cgactctaga ggatccccgg tttttgttgt gtggaattgt gagcggat 48 <210> 22 <211〉 48 1221855 10/12< 212〉 DNA < 213 > Artificial Sequence < 220> < 223 > Description of Artificial Sequence: Artificially synthesized primer sequence < 400 > 21 cgactctaga ggatccccgg tttttgtttgt gtggaattgt gagcggat 48 < 210 > 22 < 211> 48 1221855 10/12

<212〉 DNA <213> Artificial Sequence <220〉 <223〉Description of Artificial Sequence Artificially synthesized primer sequence <400> 22 tgactctaga ggatccccgg tttttgttgt gtggaattgt gagcggat< 212〉 DNA < 213 > Artificial Sequence < 220〉 < 223〉 Description of Artificial Sequence Artificially synthesized primer sequence < 400 > 22 tgactctaga ggatccccgg tttttgttgt gtggaattgt gagcggat

<210> 23 <211> 47 <212〉 DNA <213> Artificial Sequence <220> <223〉Description of Artificial Sequence Artificially synthesized primer sequence <400> 23 cgtcgtgact gggaaaaccc tttttgtgcg ggcctcttcg ctattac <210> 24 1 <211〉 21 <212〉 DNA · <213> Artificial Sequence <220> <223> Description of Artificial Sequence .-Artificially synthesized primer sequence - <400> 24 actttatgct tccggctcgt a< 210 > 23 < 211 > 47 < 212> DNA < 213 > Artificial Sequence < 220 > < 223〉 Description of Artificial Sequence Artificially synthesized primer sequence < 400 > 23 cgtcgtgact gggaaaaccc tttttgtgcg ggcctcttcg ctattac < 210 &; 24 1 < 211〉 21 < 212〉 DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence .-Artificially synthesized primer sequence-< 400 > 24 actttatgct tccggctcgt a

<210> 25 <211> 17 <212〉 DNA 48< 210 > 25 < 211 > 17 < 212> DNA 48

4747

21 1221855 11/12 <213〉 Artificial Sequence <220〉 <223> Description of Artificial Sequence-.Artificially synthesized primer sequence <400> 25 gttgggaagg gcgatcg 1721 1221855 11/12 < 213〉 Artificial Sequence < 220〉 < 223 > Description of Artificial Sequence-.Artificially synthesized primer sequence < 400 > 25 gttgggaagg gcgatcg 17

<210〉 26 <211> 44 <212> DNA <213> Artificial Sequence <220〉 <223> Description of Artificial Sequence:Artificially synthesized primer sequence <400> 26 tgttcctgat gcagtgggca gctttagtct gcggcggtgt tctg 44< 210> 26 < 211 > 44 < 212 > DNA < 213 > Artificial Sequence < 220> < 223 > Description of Artificial Sequence: Artificially synthesized primer sequence < 400 > 26 tgttcctgat gcagtgggca gctttagtct gcggcggtgt tctg 44

<210> 27 <211> 45 <212〉 DNA <213〉 Artificial Sequence · <220> <223〉 Description of Artificial Sequence:Artificially synthesized primer sequence <400> 27 tgctgggtcg gcacagcctg aagctgacct gaaatacctg gcctg 45< 210 > 27 < 211 > 45 < 212〉 DNA < 213〉 Artificial Sequence < 220 > < 223〉 Description of Artificial Sequence: Artificially synthesized primer sequence < 400 > 27 tgctgggtcg gcacagcctg aagctgacct gaaatacctg gcctg 45

<210> 28 <211〉 18 <212> DNA <213> Artificial Sequence <220〉 <220〉1221855 12/12 <223> Description of Artificial Sequence:Artificially synthesized primer sequence <400> 28 tgcttgtggc ctctcgtg< 210 > 28 < 211〉 18 < 212 > DNA < 213 > Artificial Sequence < 220〉 < 220〉 1221855 12/12 < 223 > Description of Artificial Sequence: Artificially synthesized primer sequence < 400 > 28 tgcttgtggc ctctcgtg

<210〉 29 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:Artificially synthesized primer sequence <400> 29 gggtgtggga agctgtg< 210> 29 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of Artificial Sequence: Artificially synthesized primer sequence < 400 > 29 gggtgtggga agctgtg

Claims (1)

1 · 一種核酸的增幅方法，包括： A)提供一模板，含有（i)3，末端，包括位於3，端之第 二區域以及第一互補區域，在適當條件下可互相黏合以形 成第一環狀構造，（ii)5，末端，包括位於5，端之第二區域 ^及第二互補區域’在適當條件下可互相黏合以形成第二 環狀構造，以及（iii)連接3,端與5，端之區域； B )經由具有股替代（s t r a n d d i s p 1 a c e m e n t )活性之聚 e酉母延伸模板之3端’當第一區域與第一互補區域互相黏 合以形成第一環狀構造時，形成一模板延伸，其包括位於 3端之第三區域以及第三互補區域，其分別實質上相同於 第二互補區域與第二區域，以及其在適當條件下可互相黏 合以形成第三環狀構造； C)於延伸模板之第一環狀構造黏合一募核苷酸引 子，該引子包括位於3，端之核苷酸序列互補於第一環狀構 造之部分以及位於5,端之核苷酸序列互補於該模板之第一 區域； )經由具有股取代活性之聚合酶，順著該延伸的模 板延伸募核苷酸引子，以形成互補於該模板之新模板，由 此取代在Β) 延伸步驟形成之該模板延伸； Ε)經由具有股取代活性之聚合酶，再度延伸該延伸 模板之3端至該延伸模板之5端’當第三區域與第二互補 區域互相黏合以形成第三環狀構造時，由此以該新模板取 代該延伸模板；以及 F)重複步驟A) _ Ε) ’並採用違新模板作為步驟a )之模 板，由此增幅出該核酸。 、 1221855 月 M31 89inBfiR« 曰 修正 六、申請專利範圍 其中該延伸 其中該融解 其中該反應 其中該步驟 反岸2得請專利範圍第1項所記载的方法 反應係在融解溫度調整劑存在的狀態下進 ' ^如申請專利範圍第2項所記載的 溫度調整劑為甜菜碱。 〆 Γ J j申請專利範圍第3項所記載的方法 液令存在0.2-3.Ο Μ的甜菜碱。 5·如申請專利範圍第1項所記載的方法 A)包括· 今第A」)二合t寡核苷酸引子至—樣品單股核酸分子， ;^ 括3,端部☆，其黏合至樣品單股核 酸分子任意區域之核脊酸列；"上㈣於樣品單股核 —A2)採用適當聚合酶，由第一募核苷酸引子之3， 行延伸反應以形成第一單股核酸分子，发勺 … 樣品單股核酸分子之至少一部分之區域7二1互_1於 區，其包括第一寡核苷酸引子之5,端部分.11 —5端 = 一！股核酸分子取代樣：單股核酸分子； 4)钻σ第一养核甘酸引子至該第—單股核酸分 該第二寡核苷酸引子包括3’端部分，其黏合至樣品 酸分子，以及5’端部分，豆包括實當卜士 ， ^ 舻八；杠立ρ β 2Λ ：: 只貝上相同於樣品單股核 酸勿子任思區域之核甘酸序列； Α5)採用適當聚合酶，由第二募核苷酸引子之3，端進 行延伸反應以形成該模板； ❿ Α6)以該模板取代該第一單股核酸分子。 6·如申請專利範圍第5項所記載之方法，其中用於 2125-3116-PFl.ptc 第68頁 1221855 修正 «9106688 六、申請專利範圍 A1 )黏合步驟之第_宣# — ^ 二寡核苍酸引早後士 甘引子或用於A4)黏合步驟之第 子。 糸相同於用於C )黏合步驟之募核苷酸引 括 種於樣中偵測標的核昏酸序列的方法，包 該模拓t蛀二二專利範圍第1項所記載的增幅方法’其中 亥核板包括該標的核苷酸序列，以及 中。確〜疋否4才不的核脊酸序列存在於該增幅方法之產物 其中該確 8. 如申睛專利範圍第7項所# # aa 士、本 認步驟包括： 固弟’項所圮載的方法’ 將增幅方法之產物鱼冬古 環狀I $ t 有補於第一環狀構造或第二 攝圮之核甘酸序列的探針結人· 觀察是否探針與增幅方土 ° 及 9. 如申請專利範圍第產物間發生雜交情形。 以板子標定，且該觀察步 記載的方法…該探針 偵測是否發生凝集反應。 _ 1 〇 .如申請專利範圍第7馆α 巾田方法係於核酸檢出劑的存在己載的方法，其中該增 括： 下進行且其中該確認步驟包 觀察基於檢出劑之訊息變化。 11 · 一種方法，包括： Α)提供一模板，含有（丨彳 端之第一區域以及第一互補區末端部分，包括位於3，1. A method for amplifying a nucleic acid, comprising: A) providing a template containing (i) 3, a terminal, including a second region at the 3, and a first complementary region, which can be adhered to each other under appropriate conditions to form a first The ring structure, (ii) 5, the end, including the second region ^ and the second complementary region 'at the 5, end can be adhered to each other to form a second ring structure under appropriate conditions, and (iii) is connected to the 3, end And 5, the region of the end; B) extending the 3 ends of the template through a poly-e-mother with a strand displacing activity, when the first region and the first complementary region are bonded to each other to form a first ring structure, A template extension is formed, which includes a third region and a third complementary region at the 3 ends, which are substantially the same as the second complementary region and the second region, respectively, and can be adhered to each other to form a third loop under appropriate conditions. Structure; C) A nucleotide primer is adhered to the first circular structure of the extension template. The primer includes a nucleotide sequence at the 3 end that is complementary to the first circular structure and a nucleoside at the 5 end. The sequence is complementary to the first region of the template;) through a polymerase having a strand substitution activity, nucleotide primers are extended along the extended template to form a new template complementary to the template, thereby replacing the extension in B) The template is formed by step extension; Ε) through a polymerase having a strand substitution activity, extending the 3 end of the extension template to the 5 end of the extension template again when the third region and the second complementary region are adhered to each other to form a third loop During the structure-like construction, the new template is used to replace the extended template; and F) the steps A) _E) ′ are repeated and the new template is used as the template of step a), thereby amplifying the nucleic acid. 、 1221855 month M31 89inBfiR «Amendment VI. The scope of patent application where the extension is in which the melting is in the reaction where the step is reversed. The method described in item 1 of the patent scope is in the state where the melting temperature adjuster is present. Downward: ^ The temperature regulator described in item 2 of the scope of patent application is betaine. 〆 Γ J j The method described in item 3 of the scope of application for a patent has a betaine of 0.2 to 3.0 μM. 5. Method A) as described in item 1 of the scope of patent application, including the present A ") dinucleotide t-oligonucleotide primer to a sample single-stranded nucleic acid molecule; ^ including 3, the end ☆, which adheres to Nucleic acid column in any region of the single-stranded nucleic acid molecule of the sample; " Listed to the single-stranded core of the sample-A2) Using an appropriate polymerase, an extension reaction is performed from 3 of the first nucleotide primers to form the first single-stranded nucleic acid molecule. Nucleic acid molecules, hair scoops ... At least part of the region of the sample single-stranded nucleic acid molecule 7 2 1 _1 region, which includes the 5, end portion of the first oligonucleotide primer. 11-5 end = one! Stranded nucleic acid molecule substitution sample: single-stranded nucleic acid molecule; 4) drill σ first nucleotide primer to the first-single-stranded nucleic acid point, the second oligonucleotide primer includes a 3 'end portion, which is adhered to the sample acid molecule, And the 5 'end part, the bean includes a real bashi, ^ 舻 ；; the bar ρ β 2Λ :: only the same nucleotide sequence as the sample single-stranded nucleic acid bezirensi region; Α5) using an appropriate polymerase, The 3rd end of the second nucleotide-priming primer is subjected to an extension reaction to form the template; ❿ A6) The template is used to replace the first single-stranded nucleic acid molecule. 6. The method as described in item 5 of the scope of patent application, which is used in 2125-3116-PFl.ptc page 68 1221855 Amend «9106688 6. Application scope of patent A1) The first step of the bonding step _ Xuan # — ^ Second oligo Cyanamic acid is used to induce early and post-sweetening primers or to be used in the A4) bonding step.糸 Same as the method used in the C) adhesion step to include the method of detecting the target nucleotide sequence in the sample, including the amplification method described in item 1 of the scope of patent 22 The Haihe nuclear plate includes the target nucleotide sequence as well as the middle. True ~ No No 4 The nucleotide sequence exists in the product of the amplification method, which should be true. 8. As mentioned in the patent scope of the 7th item # # aa, the recognition steps include: Method 'The product of the amplification method Yu Donggu ring I $ t has a probe that complements the nucleotide sequence of the first ring structure or the second photograph. · Observe whether the probe and the increase angle are 9 ° . If hybridization occurs between products in the scope of the patent application. Calibrate with a plate, and the method described in this observation step ... The probe detects whether an agglutination reaction has occurred. _ 1. For example, if the α-field method of the 7th pavilion of the scope of patent application is based on the existence of a nucleic acid detection agent, the method includes the following: and the confirmation step includes observing a change in the information based on the detection agent. 11 · A method comprising: A) providing a template comprising a first region at the (丨 彳 end) and a terminal portion of the first complementary region, including the third region, 2ΐ25·3ΐΐ6·ρρι Ptc 第69頁 形成第一環狀構造，（i U Υ，在適當條件下可互相黏 末端部分，包括位於5, ^218552ΐ25 · 3ΐΐ6 · ρρι Ptc p.69 Form the first ring structure, (i U Υ, can be sticky to each other under appropriate conditions, the terminal part, including the ^ 21855 ^____89106688 六、申請專利範圍 、之弟二區域以及弟一互補區域，在適當條件下可互相黏 合以形成第二環狀構造’以及（i i i )連接3，端部分與5，端 部分之區域； B)經由具有股取代活性之聚合酶由模板之3,端延伸 至5 ，當第一區域與第一互補區域互相黏合以形成第一琿 狀構造時，形成一模板延伸，其包括位於3 ’端之第三區域 ，及第三互補區域’其分別實質上相同於第二互補區域^ 第二區域，以及其在適當條件下可互相黏合以形成第三产 狀構造； 一衣 C)於延伸模板之第一環狀構造黏合一募核苷酸引 子’該引子包括位於3 ’端之核苷酸序列互補於第一環狀構 &之部分以及位於5 ’端之核苷酸序列互補於該模板之第一 區域； D)經由具有股取代活性之聚合酶，順著該延伸的模 板延伸該寡核苷酸引子，以形成互補於該模板之新模板； 以及 ' E)以該新模板取代該延伸模板。 12.如申請專利範圍第11項所記載之方法，其中該新 模板具有（i ) 3 ’端部分，包括第二區域與第二互補區城， 位於3 ’端，在適當條件下互相黏合以形成第二環狀構造， (ii) 5端部分，包括第一區域與第一互補區域，位於5’ 端，在適當條件下互相黏合以形成第一環狀構造，以及 (1 i i)連接該3 ’端部分與該5 ’端部分之部分，該方法更包 括： F) 經由I右股取夜活性之綮么_，π你妓新捃此么^ ____ 89106688 6. The scope of the patent application, the second area and the first complementary area can be bonded to each other under appropriate conditions to form a second ring structure 'and (iii) the area connecting 3, end portions and 5, end portions; B) extends from the 3, end of the template to 5 via a polymerase having a strand substitution activity. When the first region and the first complementary region are adhered to each other to form a first ridge-like structure, a template extension is formed, which includes a 3 ' The third region and the third complementary region are respectively substantially the same as the second complementary region ^ the second region, and they can be adhered to each other under appropriate conditions to form a third occurrence structure; The first circular structure of the template is bonded to a nucleotide primer. The primer includes a nucleotide sequence at the 3 'end that is complementary to the first circular structure & and a nucleotide sequence at the 5' end that is complementary to A first region of the template; D) extending the oligonucleotide primers along the extended template via a polymerase having strand substitution activity to form a new template complementary to the template; and 'E) to The new template replaces the extended template. 12. The method as described in item 11 of the scope of patent application, wherein the new template has a (i) 3 'end portion, including a second region and a second complementary region, located at the 3' end and bonded to each other under appropriate conditions to Forming a second ring structure, (ii) a 5-terminal portion, including a first region and a first complementary region, located at the 5 'end, adhering to each other under appropriate conditions to form a first ring structure, and (1 ii) connecting the The 3 'end part and the 5' end part, the method further includes: F) Take the right of the night activity via I right strand_, π you prostitute new? 2125-3116-PFl.ptc 第70頁 1221855 ---案號 89106688_年月日__^__ 六、申請專利範圍 · 3’端至該新模板之5’端，當第二區域與第二互補區域互相 黏合以形成第二環狀構造時，形成一模板延伸，包括第三 區域與第三互補區域，其係分別實質上相同於第一互補區 域與第一區域，且在適當條件下互相黏合以形成第三環狀 構造； G) 將第二募核苷酸引子黏合至該延伸新模板之第二 環狀構造，上述第二寡核普酸引子包括位於3，端之核苔酸 序列互補於第二環狀構造之部分以及位於5，端之核苷酸序 列互補於該模板之第二互補區域； H) 經由具有股取代活性之聚合酶，順著該新延伸模 板延伸該第二寡核苷酸引子，以形成第三模板，該第三模 板係實質上相同於該模板。 13·如申請專利範圍第1 2項之方法，更包括： J)以該第三模板取代該新模板。 14·如申請專利範圍第11項之方法，更包括： 採用該第三模板重複步驟B)至步驟I )。 15·如申請專利範圍第11項之方法，其中D)之延伸+ 驟取代該模板延伸之序列，該模板延伸係互補於該模板^ 端部分，以允許該第三區域與第三互補區域互相黏合以 成該第三環狀構造，該方法更包括： > 再度由該延伸模板之3,端延伸至該模板之5，端， 取代步驟E)之新模板。 此 16.如申請專利範圍第1 5項之方法，更包括： 以第二募核苷酸引子黏合至該第三環狀構造，上述第 寡核昔酸引子包括位於3,端之核脊酸序列互補於第三-2125-3116-PFl.ptc Page 70 1221855 --- Case No. 89106688_year month __ ^ __ VI. Scope of patent application 3 'end to the 5' end of the new template, when the second area and the second When the complementary regions are bonded to each other to form a second ring structure, a template extension is formed, including a third region and a third complementary region, which are substantially the same as the first complementary region and the first region, respectively, and are mutually compatible under appropriate conditions. Gluing to form a third loop structure; G) gluing a second nucleotide primer to the second loop structure of the extended new template, the second oligonucleotide primer includes a nucleotide sequence at the 3 end The portion complementary to the second circular structure and the nucleotide sequence at the 5 ′ end are complementary to the second complementary region of the template; H) The second extension template is extended by a polymerase having strand substitution activity along the new extension template Oligonucleotide primers to form a third template, which is substantially the same as the template. 13. The method of claim 12 in the scope of patent application, further comprising: J) replacing the new template with the third template. 14. The method according to item 11 of the patent application scope, further comprising: repeating steps B) to I) using the third template. 15. The method according to item 11 of the patent application, wherein the extension of D) + step replaces the sequence of the template extension, and the template extension is complementary to the ^ -end portion of the template to allow the third region and the third complementary region to mutually Bonding to form the third ring structure, the method further includes: > Extending from the 3 end of the extension template to the 5 end of the template again, replacing the new template of step E). 16. The method according to item 15 of the scope of patent application, further comprising: adhering to the third loop structure with a second nucleotide-priming primer, and the above-mentioned oligonucleotide primer includes a ribonic acid at the 3 terminal The sequence is complementary to the third- 12218551221855 1221855 ___案號 89106688 _年 月 日 修正 六、申請專利範圍 採用適當之聚合酶，由該第二寡核苷酸引子之3，端進 行延伸反應，以形成第二單股核酸分子，其包括（i ) 3，端 部分’互補於該第一單股核酸分子之5，端部分，該3，端部 分包括位於3端之第一區域與第一互補區域，在適當條件 下可互相黏合而形成第一環狀構造，以及（i丨）5，端部 包括位於5 &之苐二互補區域與第二區域，在適當條 件下可互相黏合而形成第二環狀構造；以及 以該第二單股核酸分子取代該第一單股核酸分子，由 此遠第二單股核酸分子係視為在3 ’端與5，端皆具有柄狀構 造與環狀構造。 21.如申請專利範圍第2 0項所記載之方法，其中每一 延伸反應係採用具有股取代活性之聚合酶進行。 2 2·如申請專利範圍第2 0項所記載之方法，其中該以 弟一單股核酸分子取代該樣品單股核酸分子係包括： 黏合第三募核苷酸引子至該樣品單股核酸分子之3， 端’其係第一寡核苷酸引子黏合處；以及 採用具有股取代活性之聚合酶，由該第三寡核苷酸引 子之3端進行延伸反應’以該第一單股核酸分子取代該樣 品單股核酸分子。 2 3.如申請專利範圍第2 0項所記載之方法，其中該以 第一單股核酸分子取代該第一單股核酸分子係包括： 黏合第四募核苷酸引子至該第一單股核酸分子之3， 端，其係第二募核苷酸引子黏合處；以及 採用具有股取代活性之聚合酶，由該第四寡核苷酸引 子之3,端進行延伸反應，以該第二單股核酸分子取代該第1221855 ___Case No. 89106688 _ Amendment Date 6. The scope of the patent application adopts an appropriate polymerase, and the 3 end of the second oligonucleotide primer is extended to form a second single-stranded nucleic acid molecule, which includes (I) 3, the terminal portion is complementary to the 5, terminal portion of the first single-stranded nucleic acid molecule, and the 3, terminal portion includes the first region and the first complementary region at the 3 terminal, which can be adhered to each other under appropriate conditions. Forming a first ring structure, and (i 丨) 5, the end portion including the second complementary region and the second region located at 5 & can be adhered to each other under appropriate conditions to form a second ring structure; and Two single-stranded nucleic acid molecules replace the first single-stranded nucleic acid molecule, and thus the far second single-stranded nucleic acid molecule is regarded as having a stalk-like structure and a ring structure at both the 3 ′ end and the 5 end. 21. The method as described in item 20 of the scope of patent application, wherein each extension reaction is performed using a polymerase having a strand substitution activity. 2 2. The method as described in item 20 of the scope of patent application, wherein the replacement of the sample single-stranded nucleic acid molecule with a single-stranded nucleic acid molecule includes: binding a third nucleotide-priming primer to the sample single-stranded nucleic acid molecule No. 3, the terminus is the binding site of the first oligonucleotide primer; and a polymerase with strand substitution activity is used to extend the reaction from the 3 ends of the third oligonucleotide primer to the first single-stranded nucleic acid. The molecule replaces the single-stranded nucleic acid molecule of the sample. 2 3. The method described in item 20 of the scope of patent application, wherein replacing the first single-stranded nucleic acid molecule with a first single-stranded nucleic acid molecule includes: adhering a fourth nucleotide-priming primer to the first single-stranded nucleic acid molecule The 3rd end of the nucleic acid molecule is the second nucleotide primer binding site; and a polymerase with strand substitution activity is used to extend the 3rd end of the fourth oligonucleotide primer to the second end. A single-stranded nucleic acid molecule replaces the first 2125-3116-PFl,ptc 第 73 頁 1221855 案號 8910fiftRS 修正 六、申請專利範圍 一單股核酸分子。 24·如申請專利範圍第2〇項 取代步驟係經由熱變性進行。 。己載之方法，，、中母一 25·如申請專利範圍第2〇項 品單股核酸分子係RNA，且延伸該：載^方法’八中該樣 由具有反轉錄酶活性的酵素進行'弟一寡核甘酸引子係經 26·如申請專利範圍第20項所記載的方法，其中每 延伸反應係在融解溫度調整劑存在的：能下進行：^母^ 27·如申請專利範圍第26項所記制方法，其中該融 解温度調整劑為甜菜碱。 峨 28.如申請專利範圍第27項所記載的方法’其中存在 0·2-3·0 Μ的甜菜碱。 2 9 ·如申請專利範圍第2 0項所記載的方法，其中該第 一單股核酸分子之取代係始自該第一募核苷酸引子之3，端 部分黏合至該樣品單股核酸分子以及延續至該第一單股核 酸分子之至少與該第二寡核脊引子黏合之部分。 3 0·如申請專利範圍第2 0項所記載的方法，其中該第 二單股核酸分子之取代係始自該第二募核苷酸引子之3，端 部分黏合至該第一單股核酸分子以及延續至該第二單股核 酸分子之3,端。 人 31. —種複製核酸分子的方法，包括： Α)製備如申請專利範圍第2〇項之第二單股核酸分 子，由此形成一模板； Β)採用具有股取代活性之聚合酶，由該模板之3，端 延伸至5’端，當該第-土域與^互補區域互相黏合以形 1221855 案號 89106688 J：_Ά 曰 修- 其依序包括該第二 在適當條件下互相 造一寡核苷酸引 少互補於該第一環 序列，互補於該模 ’順著該延伸模板 成一新模板，互補 的方法，更包括： 的方法，其中D)之 延伸，以允許該第 第三環狀構造，該 模板之5’端，由此 之方法，更包括： 狀構造，上述第 序列，至少互補於 核脊酸序列，互補 伸該第 六、申請專利範圍 成第，環狀構造時，形成一模板延伸， 互補隱域與第二區域’位於3 ’端，以及 黏合以形成第三環狀構造； C) 黏合至該延伸模板之第一環狀構 子，其包括位於3 端之核甘酸序列，至 狀構造之部分，以及位於5’端之核苷酸 板之第一區域；以及 D) 採用具有股取代活性之聚合酶 進行該寡核苷酸引子之延伸反應，以形 於步驟Α)形成之模板。 32. 如申請專利範圍第3 1項所記載 Ε) 以該新模板取代該延伸模板。 33. 如申請專利範圍第32項所記載 延伸步驟取代Β)延伸步驟形成之該模板 二互補區域與第二區域互相黏合以形成 Ε)取代步驟包括： 再度由該延伸模板之3 ’端延伸至該 取代該新模板。 34·如申請專利範圍第33項所記載 以第二寡核苷酸引子黏合至該第三 二寡核苷酸引子包括位於3 ’端之核脊酸 第三環狀構造之部分，以及位於5，端之 於該模板之第三區域；以及 經由具有股取代活性之聚合酶，延 引子之3’端。 核苷酸2125-3116-PFl, ptc Page 73 1221855 Case No. 8910fiftRS Amendment 6. Scope of patent application A single-stranded nucleic acid molecule. 24. If the scope of patent application is No. 20, the substitution step is performed by thermal denaturation. . The method that has been included, and the mother-in-law 25. If the patent application scope is No. 20, the single-stranded nucleic acid molecule is RNA, and the method is extended: The method `` Eight in this method is performed by an enzyme having reverse transcriptase activity '' Di-oligonucleotide primers are described in 26. The method described in item 20 of the scope of patent application, wherein each extension reaction is performed in the presence of a melting temperature adjuster: capable of: ^ mother ^ 27. If the scope of patent application is 26 The method according to item 4, wherein the melting temperature adjuster is betaine. 28. The method described in item 27 of the scope of the patent application, wherein betaine is present in an amount of 0.2-2.0. 29. The method as described in item 20 of the scope of the patent application, wherein the substitution of the first single-stranded nucleic acid molecule starts from 3 of the first nucleotide primer, and the end portion is adhered to the single-stranded nucleic acid molecule of the sample. And continued to at least a portion of the first single-stranded nucleic acid molecule that is adhered to the second oligonucleotide primer. 30. The method as described in item 20 of the scope of patent application, wherein the substitution of the second single-stranded nucleic acid molecule starts from 3 of the second nucleotide-priming primer, and the end portion is adhered to the first single-stranded nucleic acid. Molecule and the 3 'end of the second single-stranded nucleic acid molecule. Human 31. A method for replicating a nucleic acid molecule, including: A) preparing a second single-stranded nucleic acid molecule as described in the scope of application for patent No. 20, thereby forming a template; B) using a polymerase having strand substitution activity, and The 3 'end of the template extends to the 5' end. When the-soil region and the ^ complementary region are bonded to each other in the shape 1221855 Case No. 89106688 J: _Ά Yue Xiu-It includes the second one in order to create one under appropriate conditions. Oligonucleotides are complementary to the first loop sequence, complementary to the pattern, and follow the extension template to form a new template. The method of complementation further includes: a method, wherein D) is extended to allow the third The ring structure, the 5 'end of the template, and the method further includes: a shape structure, the above-mentioned sequence is at least complementary to the nucleotide sequence, complementary to the sixth, and the scope of the patent application is the first, when the circle structure To form a template extension, where the complementary hidden domain and the second region are located at the 3 'end, and are bonded to form a third ring structure; C) the first ring structure bonded to the extension template, which includes the 3 Nucleic acid And the first region of the nucleotide plate at the 5 'end; and D) using a polymerase having a strand substitution activity to perform the extension reaction of the oligonucleotide primer to form step A ) Formed template. 32. As described in item 31 of the scope of the patent application E) Replace the extended template with the new template. 33. As described in item 32 of the scope of patent application, the extension step replaces the second complementary area of the template formed by the extension step B) and the second area adheres to each other to form E) The replacement step includes: once again extending from the 3 'end of the extension template to The new template should be replaced. 34. As described in item 33 of the scope of the patent application, the second oligonucleotide primer is bonded to the third oligonucleotide primer and includes a portion of the third loop structure of ribonic acid located at the 3 ′ end, and located at 5 To the third region of the template; and to extend the 3 'end of the primer via a polymerase having a strand substitution activity. Nucleotide 2125-3116-PFl.ptc 第75頁 1221855 _案號 891Q6688 六、申請專利範圍 年2125-3116-PFl.ptc Page 75 1221855 _Case No. 891Q6688 6. Scope of Patent Application Year 所記載的方法，更包括採 ’重複步驟B)至步驟E)。 所記載的方法，其中該延 的狀態下進行。 所記載的方法，其中該融 35.如申請專利範圍第32項 用步驟D)形成之新板板作為模板 3 6.如申請專利範圍第3 1項 伸反應係在融解溫度調整劑存在 3 7.如申請專利範圍第3 6項 解溫度調整劑為甜菜碱。 38. 如申請專利範圍第37項所記載的方法，其中該反 應液中存在0.2 - 3·0 Μ的甜菜碱。The described method further includes repeating steps B) to E). The method described is performed in a delayed state. The method described, wherein the melting 35. The new plate formed by step D) as the template 32 in the scope of the patent application 3 as the template 3 6. The extension reaction in the scope of the patent application 31 item 1 is in the presence of a melting temperature regulator 3 7 . If the patent application scope of the 36th solution temperature regulator is betaine. 38. The method according to item 37 of the scope of patent application, wherein 0.2 to 3.0 M betaine is present in the reaction solution. 39. 一種製備5’端與3’端具有柄狀構造（stem)與環狀 構造（loop)之單股核酸分子的方法，其包括： 提供第一單股核酸分子，包括5,端部分，其包括位於 5’端之第一區域以及第一互補區域，在適當條件下互相黏 合以形成一環狀構造； 黏合第一寡核苷酸引子至該第一單股核酸分子，該第 一寡核苷酸引子包括3’端部分，其黏合至該第一單股核酸 分子，以及5’端部分，其包括實質上相同於該第一單股核 酸分子之任意區域之核苷酸序列；39. A method for preparing a single-stranded nucleic acid molecule having a stem structure and a loop structure at the 5 'end and the 3' end, comprising: providing a first single-stranded nucleic acid molecule, including a 5, terminal portion, It includes a first region at the 5 ′ end and a first complementary region, which are adhered to each other under a suitable condition to form a circular structure; a first oligonucleotide primer is adhered to the first single-stranded nucleic acid molecule, and the first oligo The nucleotide primer includes a 3 'end portion that is adhered to the first single-stranded nucleic acid molecule, and a 5' end portion that includes a nucleotide sequence that is substantially identical to any region of the first single-stranded nucleic acid molecule; 採用適當之聚合酶，由該第一寡核苷酸引子之3，端進 行延伸反應，以形成第二單股核酸分子，其包括（i ) 3，端 部分，互補於該第一單股核酸分子之5，端部分，該3，端部 分包括位於3 ’端之第一區域與第一互補區域，在適當條件 下可互相黏合而形成第一環狀構造，以及（i i) 5，端部分， 包括位於5’端之第案區域以及第二互補區域，在適當條件 下可互相黏合以形成第二環狀構造； 以該第二單股核酸分子取代該第一單股核酸分子，係Using an appropriate polymerase, an extension reaction is performed from the 3 'end of the first oligonucleotide primer to form a second single-stranded nucleic acid molecule, which includes (i) 3, a terminal portion that is complementary to the first single-stranded nucleic acid The 5, end portion of the molecule, the 3, end portion includes a first region and a first complementary region located at the 3 'end, which can be adhered to each other under appropriate conditions to form a first ring structure, and (ii) a 5, end portion Including a case region and a second complementary region at the 5 ′ end, which can be adhered to each other to form a second circular structure under appropriate conditions; replacing the first single-stranded nucleic acid molecule with the second single-stranded nucleic acid molecule, 2125-3116-PFl.ptc 第76頁 1221855 案號 89106688 年2125-3116-PFl.ptc page 76 1221855 case number 89106688 始 並 分 視 伸 合 解 0· 子 延 成 第 互 成 子 狀 申請專利範圍 自該第-寡核苷酸黏合至該第—單股核酸 延續至該第二核酸分子之3,端，i#子3 &口I5刀 子取代該第一單股核酸分子時，^ ，第一單股核酸 為具有3’端㉝分形&第一環狀構‘皁m刀子係 環狀構造。 楫以及5端部分形成第 40如申請專利範圍第39項所記載之方法 反應係採用具有股取代活性之聚人 ，、T該延 41. 如中請專利範圍第39項所二# 3二 及延伸反應係在融解溫度調整劑#/ 二，其中該黏 ,n ^ d存在的狀態下進行。 42. 如申請專利範圍第4丨項所記載的方法， 溫度調整劑為甜菜碱。 /、τ θ成 43. 如申請專利範圍第42項所記載的方法，盆 2-3· 0 Μ的甜菜碱。 八r仔在 44. 一種複製核酸分子的方法，包括· A) 製備如申請專利範圍第39項之第二單股核酸 ，由此形成一模板； B) 採用具有股取代活性之聚合酶，由該模板之3， 伸至5端，當該第一區域與第一互補區域互相黏合以形 第一環狀構造時，形成一模板延伸，其包括位於3，端之 三區域以及第三互補區域，其分別實質上相同於該第二 補區域以及第二區域，以及在適當條件下互相黏合以^ 第三環狀構造； 7 c)黏合至該延伸模板之第一環狀構造一募核苷酸引 ，其包括位於3端之核苔酸序列，至少互補於該第一環 構造之部分，以及位於5,端之核苷^序列，互補於該‘Beginning and scoring the extension and extension solution. The extension of the sub-segment into the first sub-segment applies for a patent ranging from the attachment of the -oligonucleotide to the -single-stranded nucleic acid to the 3rd end of the second nucleic acid molecule, i # 子When the 3 & port I5 knife replaces the first single-stranded nucleic acid molecule, the first single-stranded nucleic acid has a 3 'terminal ㉝ fractal & first circular structure' soap m knife system ring structure.楫 And the 5 terminal part forms the method as described in item 39 of the scope of the patent application. The reaction is to use a polymer with a substitution activity, and the extension is 41. Such as the scope of the patent, the 39th place of the patent # 3 二 和The extension reaction is performed in a state where the melting temperature modifier # / II is present, and the viscosity, n ^ d is present. 42. The method described in item 4 of the patent application scope, wherein the temperature regulator is betaine. / 、 Τ θ 成 43. The method described in item 42 of the scope of patent application, pots of 2-3 · 0M betaine. Ba Rzai 44. A method for replicating a nucleic acid molecule, including: A) preparing a second single-stranded nucleic acid such as the scope of application for item 39, thereby forming a template; B) using a polymerase having strand substitution activity, The third part of the template extends to the fifth end. When the first region and the first complementary region are bonded to each other to form a first ring structure, a template extension is formed, which includes three regions at the third end and a third complementary region. , Which are substantially the same as the second complement region and the second region, and are bonded to each other under a suitable condition to form a third ring structure; 7 c) a first ring structure bonded to the extended template, a nucleoside-recruiting compound An acid primer, which includes a nucleotide sequence at the 3 terminal end that is at least complementary to the first loop structure, and a nucleotide sequence at the 5 terminal end that is complementary to the ' 2125-3116-PFl.ptc 1221855 六、申請專利範圍 板之第一區域；以及 D) 採用具有股取代活性之聚合_ — 進行該寡核苔酸引子之延伸反應，以心丨員者該延伸模板 於步驟A)形成之模板。 元成一新模板’互補 如申請專利範圍第44項所記载的方法 E) 以該新模板取代該延伸模板。 t g π · 46. 如申請專利範圍第45項所記裁 延伸步驟取代Β)延伸步驟形成之該模板 ’ ，、中）之 一 /佚奴延伸，以分妹古方楚 一互補區域與第三區域互相黏合 ° ^ Ε)取代步驟包括： 4成弟三環狀構造，該 再度由该延伸模板之3 ’端延伸$兮伊r， 取代該新模板。 板之5，端，由此 47. 如申請專利範圍第46項所記載之方法，更包括： 一以第二寡核苷酸引子黏合至該第三環狀構造，上述第 :养核甘酸引子包括位於3,端之核苷酸序列，至少互補於 二：％狀構造之部A，以及位於5,端之核苷酸序列，互補 於該模板之第三區域；以及 經由具有股取代活性之聚合酶，延伸該第二寡核苷酸 引子之3’端。 48. 如申請專利範圍第45項所記載的方法，其中該新 杈板具有(1) 5’端部分，包括第_區域與第一互補區域， 位於5’端，在適當條件下互相黏合以形成第一環狀構造， (11) 3’端部分，包括第二區域與第二互補區域，位於3， 端，在適當條件下互相黏合以形成第二環狀構造，該方法 更包括： 2125-3116-PFl.ptc 第78頁 F)經由具有股取代活性之聚合酶，延伸該新模板之 3端至該新模板之5 ’端’當第二區域與第二互補區域互相 點合以形成第二環狀構造時，形成一模板延伸，包括第= 區域與第三互補區域，其係分別實質上相同於第一互補區 域與第一區域，且在適當條件下互相黏合以形成第三環狀 構造； —< G)將苐二募核苔酸引子黏合至該延伸新模板之第二 ^狀構造，上述第二寡核苷酸引子包括位於3，端之核脊酸 序列，至少互補於第二環狀構造之部分，以及位於5，端之 核苔酸序列，互補於該模板之第二互補區域； H)經由具有股取代活性之聚合酶，順著該新延伸模 板延=該第二寡核苷酸引子，以形成第三模板，該第三模 板係貫質上相同於該模板。 49·如申請專利範圍第48項所記載之方法，更包括： ί)以該第三模板取代該新模板。 51·如申請專利範圍第44項 伸反應係在融解溫度調整劑存在 5 2·如申請專利範圍第5 1項 解溫度調整劑為甜菜碾。 5 3.如申請專利範圍第5 2項 應液中存在0.2-3.0 Μ的甜菜碱。 50·如申請專利範圍第49項所記載之方法，f句括· 採用該第三模板重複步驟B)至步驟丨）。 所記載之方法，其中該延 的狀態下進行。 所記載的方法，其中該融 所記載的方法，其中該反 一種製備5,端與3,端具有 構造（loop)之單股核酸分子的方法 54· 狀構造（stem)與環狀 其包括·· 1221855 ____案號 8910fifi88 --生— 月〜^_ 六、申請專利範圍 黏合第一寡核苷酸引子至樣品單股核酸分子，該第一 募核苷酸包括3，端部分，其黏合至該樣品單股核酸分子， 以及5 ’端部分，其包括實質上相同於該樣品單股核酸分子 之任意區域之核苷酸序列； 採用適當之聚合酶’由該第一寡核苷酸引子之3，端進 行延伸反應，以形成第一單股核酸分子，其包括5，端部 分’包括位於5 ’端之第一區域與第_互補區域，在適當條 件下可互相黏合而形成一環狀構造； 以该第一單股核酸分子取代該樣品單股核酸分子，始 自該第一寡核苷酸引子之3’端部分黏合至該樣品單股核酸 分子以及延續至至少該第一單股核酸分子可黏合至第二寡 核苷酸引子之部分； 黏合該第二寡核苷酸引子至該第一單股核酸分子，該 第二寡核脊酸包括3 ’端部分，其黏合至該第一單股核酸分 子，以及5’端部分，其包括實質上相同於該第一單股核酸 分子之任意區域之核脊酸序列； 採用適當之聚合酶，由該第二寡核苷酸引子之3，端進 行延伸反應，以形成第二單股核酸分子，其包括（丨）3，端 部分，互補於該第一單股核酸分子之5,端部分，該3,端 分包括位於3’端之第一區域與第一互補區域，在適當條件 下可互相黏合而形成第一環狀構造，以及（丨丨）5，端部 分，包括位於5’端之第二互補區域與第二區域，在適當 件下可互相黏合而形成第二環狀構造；以及 /、 >人球乐2125-3116-PFl.ptc 1221855 VI. The first area of the patent application board; and D) Polymerization with strand substitution activity is used to perform the extension reaction of the oligonucleotide primer, so that the extension template can be used by the user. The template formed in step A). Yuanchengyi's new template 'complements the method described in item 44 of the scope of patent application E) Replace the extended template with the new template. tg π · 46. If the extension step recorded in item 45 of the scope of patent application replaces the one of the template formed by the extension step B), the middle / one slave extension is used to divide the complementary area of the ancient and the ancient areas and the third area. Adhesion to each other (° E) The replacement step includes: 40% tricyclic structure, which is extended from the 3 ′ end of the extension template again to replace the new template. The 5th end of the plate, thus 47. The method as described in item 46 of the scope of patent application, further comprising: a second oligonucleotide primer is adhered to the third loop structure, the above-mentioned: nucleoside primer Including a nucleotide sequence at the 3 'end that is at least complementary to the two:%-like structure part A, and a nucleotide sequence at the 5, end that is complementary to the third region of the template; and Polymerase, extending the 3 'end of the second oligonucleotide primer. 48. The method described in item 45 of the scope of patent application, wherein the new branch plate has (1) a 5 'end portion, including a _ area and a first complementary area, which are located at the 5' end and are bonded to each other under appropriate conditions to To form a first ring structure, (11) The 3 'end portion, including the second region and the second complementary region, is located at the 3, end, and is bonded to each other under appropriate conditions to form a second ring structure. The method further includes: 2125 -3116-PFl.ptc page 78 F) Via a polymerase having a strand substitution activity, the 3 end of the new template is extended to the 5 'end' of the new template when the second region and the second complementary region meet each other to form In the second ring structure, a template extension is formed, including the third region and the third complementary region, which are substantially the same as the first complementary region and the first region, respectively, and are bonded to each other under appropriate conditions to form a third loop. -≪ G) Adhering the second dinucleotide primer to the second structure of the extended new template, the second oligonucleotide primer includes a nucleotide sequence at the 3 'end, at least complementary In the part of the second ring structure, And the nucleotide sequence at the 5 ′ end is complementary to the second complementary region of the template; H) via the polymerase with strand substitution activity, extending along the new extension template = the second oligonucleotide primer to A third template is formed, which is essentially the same as the template. 49. The method described in item 48 of the scope of patent application, further comprising: til) replacing the new template with the third template. 51. If the scope of the patent application is No. 44, the extension reaction exists in the melting temperature adjuster 5 2 If the scope of the patent application is No. 51, the temperature modifier is beet mill. 5 3. As in claim 52 of the scope of patent application, 0.2-3.0 M betaine is present in the solution. 50. The method described in item 49 of the scope of patent application, the f-sentence includes repeating steps B) to 丨) using the third template. The method described is performed in the delayed state. The method described, wherein the method described in the present invention, wherein the reverse method of preparing a single-stranded nucleic acid molecule having a loop at 5, and 3 ends, a stem structure, and a loop, including: · 1221855 ____ Case No. 8910fifi88-Health-Month ~ ^ _ VI. Patent application scope Adhesion of the first oligonucleotide primer to the single-stranded nucleic acid molecule of the sample, the first nucleotide collection includes 3, terminal portion, which adheres To the sample single-stranded nucleic acid molecule, and the 5 'end portion, which includes a nucleotide sequence substantially identical to any region of the sample single-stranded nucleic acid molecule; using an appropriate polymerase' from the first oligonucleotide primer 3, the end is extended to form a first single-stranded nucleic acid molecule, which includes 5, and the end portion includes a first region and a complementary region at the 5 'end, which can be adhered to each other to form a loop under appropriate conditions. Like structure; replacing the sample single-stranded nucleic acid molecule with the first single-stranded nucleic acid molecule, starting from the 3 'end portion of the first oligonucleotide primer adhered to the sample single-stranded nucleic acid molecule and continuing to at least the first single-strand nucleic acid molecule Stock core The molecule can be adhered to a portion of a second oligonucleotide primer; the second oligonucleotide primer is adhered to the first single-stranded nucleic acid molecule, and the second oligonucleotide comprises a 3 'end portion which is adhered to the first A single-stranded nucleic acid molecule, and a 5 'end portion, comprising a nucleotide sequence substantially identical to any region of the first single-stranded nucleic acid molecule; using a suitable polymerase, 3, the end is subjected to an extension reaction to form a second single-stranded nucleic acid molecule, which includes (丨) 3, a terminal portion that is complementary to the 5, terminal portion of the first single-stranded nucleic acid molecule, and the 3, terminal portion includes a 3 ' The first region and the first complementary region at the end can be adhered to each other to form a first ring structure under appropriate conditions, and (丨 丨) 5, the end portion includes the second complementary region and the second region at the 5 'end. , Can be bonded to each other to form a second ring structure under appropriate pieces; and /, > Renqiu 12218551221855 造與環狀構造。 μ ^ e、如申請專利範圍第54項所記載之方法，其中每一 申反應係採用具有股取代活性之聚合酶進行。 私^56·如申請專利範圍第54項所記載之方法，其中每一 -口與延伸反應係在融解溫度調整劑存在的狀態下進行。 57·如申請專利範圍第56項所記載的方法，其中該融 解溫度調整劑為甜菜碱。 58·如申請專利範圍第57項所記載的方法，其中存在 〇· 2-3· Ο Μ的甜菜碱。Construction and ring structure. μ ^ e. The method described in item 54 of the scope of patent application, wherein each application reaction is performed using a polymerase having a strand substitution activity. 56. The method described in item 54 of the scope of patent application, wherein each of the mouth-to-elongation reaction is performed in the presence of a melting temperature modifier. 57. The method according to item 56 of the application, wherein the melting temperature adjuster is betaine. 58. The method as described in claim 57 of the scope of patent application, wherein betaine of 0.2-2.0M is present. 一抑59·如申請專利範圍第54項所記載的方法，其中該第 二單股核酸分子之取代係始自該第二募核苷酸引子之3,端 部分黏合至該第一單股核酸分子以及延續至該第二單股核 酸分子之3’端。 6 0· —種複製核酸分子的方法，包括： A) 製備如申請專利範圍第5 4項之第二單股核酸分 子，由此形成一模板；Yiyi 59. The method described in item 54 of the scope of patent application, wherein the replacement of the second single-stranded nucleic acid molecule starts from the 3rd end of the second nucleotide primer and is adhered to the first single-stranded nucleic acid. The molecule and continues to the 3 'end of the second single-stranded nucleic acid molecule. 60. A method for copying a nucleic acid molecule, including: A) preparing a second single-stranded nucleic acid molecule as claimed in item 54 of the patent application, thereby forming a template; B) 採用具有股取代活性之聚合酶，由該模板之3,端 延伸至5 ’端，當該第一區域與第一互補區域互相黏合以形 成第一環狀構造時，形成一模板延伸，其包括位於3，端之 第三區域以及第三互補區域，其分別實質上相同於該第二 互補區域以及第二區域’以及在適當條件下互相黏合以形 成第三環狀構造； C) 黏合至該延伸模板之第一壞狀構造一寡核奋酸引 子，其包括位於3 ’端之核苷酸序列，至少互補於該第一環 狀構造之部分，以及位於5 ’端之核苷酸序列，互補於該模B) A polymerase having strand substitution activity is used to extend from the 3 'end to the 5' end of the template. When the first region and the first complementary region are adhered to each other to form a first ring structure, a template extension is formed, It includes a third region at the 3 end and a third complementary region, which are substantially the same as the second complementary region and the second region, respectively, and adhere to each other under appropriate conditions to form a third ring structure; C) Adhesion The first bad structure to the extension template is an oligonucleotide primer comprising a nucleotide sequence at the 3 'end, at least a portion complementary to the first circular structure, and a nucleotide at the 5' end Sequence, complementary to the module 2125-3116-PFl.ptc 第81頁 1221855 案號 89106688 六、申請專利範圍 板之第一區域；以及 D) 採用具有股取代活性之聚合酶，順著該延伸模板 進行該募核苷酸引子之延伸反應，以形成一新模板，互補 於步驟A)形成之模板。 61·如申請專利範圍第6 0項所記載的方法，更包括： E) 以該新模板取代該延伸模板。 6 2·如申請專利範圍第6 1項所記載的方法，其中d )之 延伸步驟取代B)延伸步驟形成之該模板延伸，以允許該第 三互補區域與第三區域互相黏合以形成第三環狀構造，該 E)取代步驟包括： 再度由該延伸模板之3’端延伸至該模板之5，端，由此 取代該新模板。 6 3.如申請專利範圍第6 2項所記載之方法 以第二寡核脊酸引子黏合至該第三環狀構造，上述第 二寡核苷酸引子包括位於3’端之核苷酸序列’至少互補於 第三環狀構造之部# ’以及位於5’端之核苔酸序列，互補 於該模板之第三區域；以及 延伸該第二寡核苷酸 經由具有股取代活性之聚合酉每 引子之3’端。2125-3116-PFl.ptc Page 81 1221855 Case No. 89106688 6. The first area of the patent application board; and D) Using a polymerase with strand substitution activity, follow the extended template to carry out the nucleotide recruitment primer The reaction is extended to form a new template, which is complementary to the template formed in step A). 61. The method described in item 60 of the scope of patent application, further comprising: E) replacing the extended template with the new template. 62. The method as described in item 61 of the scope of patent application, wherein the extension step of d) replaces the template extension formed by the extension step B) to allow the third complementary region and the third region to adhere to each other to form a third The ring structure, the E) replacement step includes: once again extending from the 3 'end of the extension template to the 5' end of the template, thereby replacing the new template. 6 3. The method described in item 62 of the scope of the patent application is adhered to the third circular structure with a second oligonucleotide primer, and the second oligonucleotide primer includes a nucleotide sequence at the 3 'end. 'At least complementary to the third circular structure of the part #' and the nucleotide sequence at the 5 'end is complementary to the third region of the template; and extending the second oligonucleotide via a polymer having a strand substitution activity. 3 'end of each primer. 64.如申請專利範圍第6〇 j盲所# # ^ ^ ^ 间不〇 u項所，己载的其中該新 模板具有（i) 5’端部分，包括篦一 F a t < /Sr 丄 匕枯弟一區域與第一皂補區域， 位於5’，端山’在適當條'下互相黏合以形成環狀構造， (11) 3 #部分’包括第二區域與第二 ，位於3’ 端，在適當條件下互相黏合以开彡忐筮 ,^ χ ^ ^ 乂形成弟二環狀構造，該方法 更包括·64. As in the scope of the application for patent No. 60〇 blind office # # ^ ^ ^ ical office, the new template has been included therein (i) 5 'end portion, including 篦 F at < / Sr 丄The first area and the first soap area are located at 5 ', and Duanshan is bonded to each other under a suitable strip to form a ring structure. (11) 3 #Part' includes the second area and the second, located at 3 ' Under the appropriate conditions, they are bonded to each other to open the 彡 忐 筮, ^ χ ^ ^ 乂 to form a di-bicyclic structure. The method further includes · 1221855 --意號 89106688 ^. . ..±_L 修正___-^ -- 六、申请專利範圍 F)經由具有股取代活性之聚合酶，延伸該新模板之 3’端至該新模板之5，端，當第二區域與第二互補區域互相 黏合以形成第二環狀構造時，形成一模板延伸，包括第三 區域與第三互補區域，其係分別實質上相同於第一互補區 域與第一區域，且在適當條件下互相黏合以形成第三環狀 構造； G) 將第二寡核脊酸引子黏合至該延伸新模板之第二 壤狀構造，上述第二寡核苷酸引子包括位於3，端之核脊酸 序列’至少互補於第二環狀構造之部分，以及位於5，端之 核苷酸序列，互補於該模板之第二互補區域； H) 經由具有股取代活性之聚合酶，順著該新延伸模 板k伸σ亥第二募核zgr酸引子’以形成第三模板，該二、 板係實質上相同於該模板。 —核 65 I)66 採 67 伸反應 68 解溫度 69 應液中 70 第 卻甲請專 以該第三模板取代該新模板£ •如申請專利範圍第6 5項所記 用该第三模板重複步驟B)至步 •如申請專利範圍第6 0項所記 係在融解溫度調整劑存在的狀 •如申請專利範圍第67項所記 調整劑為甜菜碱。 .如申請專利範圍第6 8項所記 存在0.2-3·0 Μ的甜菜碱。 .一種套組，包括： 載之方法’更包括 載之 驟I) 載之 態下 載的 方法’更包括： 方 進 方 法 行 法 其中該延 其中該融 載的方法，其 中该反 寡核有1酸引子，包括i)3’端 核脊 其勒合1221855 --Issue 89106688 ^. .. ± _L Amendment ___- ^-VI. Patent application scope F) Extend the 3 ′ end of the new template to 5 of the new template via a polymerase with strand substitution activity At the end, when the second region and the second complementary region are adhered to each other to form a second ring structure, a template extension is formed, including the third region and the third complementary region, which are respectively substantially the same as the first complementary region and The first region, and adhere to each other under appropriate conditions to form a third circular structure; G) the second oligonucleotide primer is adhered to the second soil-like structure of the extended new template, the second oligonucleotide primer Including a nucleotide sequence at the 3 ′ end that is complementary to at least a portion of the second circular structure, and a nucleotide sequence at the 5 ′ end that is complementary to the second complementary region of the template; H) via a strand substitution activity The polymerase follows the new extension template k, and extends the second nuclear promoter zgr acid primer 'to form a third template. The two and plate systems are substantially the same as the template. —Nuclear 65 I) 66 Minute 67 Extension reaction 68 Solution temperature 69 In solution 70 No. 1 Please replace the new template with the third template. • Repeat with the third template as described in item 65 of the scope of patent application Step B) to step • As described in Item 60 of the scope of the patent application, the state of the melting temperature adjuster is present. • As described in Item 67 of the scope of the patent application, the regulator is betaine. Betaine is present in the range of 0.2 to 3.0 μM as described in item 68 of the scope of patent application. A set comprising: a method of downloading, further including a step of downloading I) a method of downloading in a state of downloading, further comprising: a method of forward method, wherein the method of delaying the fusion, wherein the anti-oligonucleotide has 1 Acid primers, including i) 3 'terminal nuclear spine 2125-3116-PFl.ptc 第83頁 1221855 案號 89106688, 一月 曰 修正 六 至 酸 端 域 子 端 採 為 第 該 之 檢 黏 股 申請專利範圍 樣品單股核酸分子並作為合成之始，以合成第一單股校 分子，其至少互補於該樣品單股核酸分子，以及（i 核苔酸序列，其互補於該第一單股核酸分子之彳壬意1區 ， 第二寡核苷酸序引子，包括黏合至該樣品單股核> 之核苷酸序列，其位於該第一募核苷酸引子黏合分 ， 第二养核有1酸引子’包括i ) 3，端核苔酸序列，勒人 用第一募核苷酸引子製備之該第一單股核酸分子，二至 合成之始，以合成第二單股核酸分子，其至少互補於， 一單股核酸分子，以及i i) 5，端核苷酸序列，其㊆忒 第二單股核酸分子之任意區域； 於 DNA聚合@母’其具有股取代活性；以及 用於DNA聚合酶延伸引子之一或多個核苷酸。 71. 如申請專利範圍第7〇項所記載的套組，更包 第四寡核甘酸引子，包括黏合至該第一單股核酸分 核苷酸序列，位於供該第三寡核苷酸引子黏合之3，子 72. 如巾請專利範圍第7〇項所記載的套組，更包括: 用以積測採用該套組其他成份進行核酸合的 出劑。 乃的 7 3· 一種合成核酸分子之方法，包括·· A)將以下成f與作為模板之樣品核酸混合·· 第-寡核苷酸引子，包括i) 3，端核苷酸序列 合至樣品單股核酸分子並作為合成之始，以合成第一/、 核酸分子’其至少_部分互補於該樣品單股核酸分子，以2125-3116-PFl.ptc, page 83, 1221855, case number 89106688, January, amended the six-to-acid end domain sub-ends to be taken as the first inspection of the application of the patent application scope sample single-stranded nucleic acid molecules and as the beginning of synthesis to synthesize A first single-stranded molecule, which is at least complementary to the single-stranded nucleic acid molecule of the sample, and (i) a nucleotide sequence that is complementary to the first non-nucleic acid region of the first single-stranded nucleic acid molecule, and a second oligonucleotide sequence Primer, including a nucleotide sequence bonded to the single-core nuclei of the sample, which is located in the first nucleotide primer primer binding element, the second nucleus has an acid primer 'including i) 3, a terminal nucleic acid sequence , The first single-stranded nucleic acid molecule prepared by Leren with a first nucleotide primer, two to the beginning of synthesis, to synthesize a second single-stranded nucleic acid molecule, which is at least complementary to, a single-stranded nucleic acid molecule, and ii) 5. A terminal nucleotide sequence, which is an arbitrary region of the second single-stranded nucleic acid molecule; it has strand substitution activity in DNA polymerization @ 母 '; and is used for one or more nucleotides of a DNA polymerase extension primer. 71. The kit described in item 70 of the scope of the patent application, further comprising a fourth oligonucleotide primer, including a first single-stranded nucleic acid nucleotide sequence, which is located for the third oligonucleotide primer Adhesive No. 3, Sub 72. If the kit is described in item 70 of the patent scope, the kit further includes: an agent for accumulating nucleic acid using other components of the kit. No. 7 3 · A method for synthesizing nucleic acid molecules, including ... A) Mixing the following f with the sample nucleic acid as a template ... The -oligonucleotide primer, including i) 3, the terminal nucleotide sequence is combined to The sample single-stranded nucleic acid molecule is used as the beginning of the synthesis to synthesize the first nucleic acid molecule, which is at least partially complementary to the sample single-stranded nucleic acid molecule, to 2125-3116-PFl.ptc 第84頁 12218552125-3116-PFl.ptc Page 84 1221855 __案號 89106688 六、申請專利範圍 及Gi) 5,端核旮酸序列’其互補於該第一單股核酸分子 之任意區域； 第-广核苷I序弓丨子’包括黏合至該樣品單股核酸 /刀子之核奋酸序列，其位於供該第一寡核奋酸引子黏合之 3,端； /第三寡核苷酸引子，包括i) 3,端核苷酸序列，黏合 至採用第一寡核苷酸引子製備之該第一單股核酸分子，並 作為合成之始，以合成第二單股核酸分子，其至少部分互 補於該第一單股核酸分子，以及i i ) 5,端核苷酸序列，其 互補於該第二單股核酸分子之任意區域； 第四寡核苷酸引子，包括黏合至該第一單股核酸分 子之核苷酸序列，位於供該第三寡核苷酸引子黏合之3， 端； DNA聚合酶，其具有股取代活性；以及 用於^NA聚合酶延伸引子之一或多個核苷酸；以及 # 一 培_育该混合物於一溫度，該溫度使構成該第一與 ，二寡核苷酸引子之核苷酸序列可以與該模板形成穩定鹼 所記載之方法，其中該混 所§己載的方法，其中該融 所記載的方法，其中該存 74·如申請專利範圍第73項 合物更包括融解溫度調整劑。 75·如申請專利範圍第74項 解溫度調整劑為甜菜碱。 76·如申請專利範圍第75項 在0·2-3·0 Μ的甜菜碱。__ Case No. 89106688 VI. Patent Application Scope and Gi) 5, The terminal nucleotide sequence 'is complementary to any region of the first single-stranded nucleic acid molecule; Sample single-stranded nucleic acid / knife nucleotide sequence, which is located at the 3 end of the first oligonucleotide primer; / Third oligonucleotide primer, including i) 3, terminal nucleotide sequence, adhesion To the first single-stranded nucleic acid molecule prepared by using the first oligonucleotide primer, and as a start of synthesis, to synthesize a second single-stranded nucleic acid molecule, which is at least partially complementary to the first single-stranded nucleic acid molecule, and ii) 5, a terminal nucleotide sequence that is complementary to any region of the second single-stranded nucleic acid molecule; a fourth oligonucleotide primer, including a nucleotide sequence that is adhered to the first single-stranded nucleic acid molecule, located at the first The three ends of the tri-oligonucleotide primer adhesion; DNA polymerase, which has strand substitution activity; and one or more nucleotides for ^ NA polymerase extension primer; and Temperature, the temperature makes the first and second oligonucleotide primers The nucleotide sequence can form a stable base with the template, wherein the method described in § IX has been included, and the method described in Fusion, wherein the deposit 74. If the 73rd composition of the scope of the patent application includes Melting temperature regulator. 75. If the scope of patent application is 74, the solution temperature adjusting agent is betaine. 76. If the scope of application for patent No. 75 is betaine at 0.2-2.0M. 2125-3116-PFl.ptc 第85頁 1221855 _案號 89106688 六、申請專利範圍 年 月 曰 修正 合物更包括： 用/以偵測A)混合步驟與B)培育步驟所形成之產物的檢 出劑。 78.如申請專利範圍第73項所記載的方法，其中該樣 品核酸係RNA，且該DNA聚合酶具有反轉錄酶活性。2125-3116-PFl.ptc Page 85 1221855 _ Case No. 89106688 Sixth, the scope of application for patent amendments include: Use / to detect the detection of products formed in A) mixing step and B) incubation step Agent. 78. The method according to claim 73, wherein the sample nucleic acid is RNA, and the DNA polymerase has reverse transcriptase activity. 2125-3116-PFl.ptc 第86頁2125-3116-PFl.ptc Page 86