TW200912307A - High density molecular alignment of nucleic acid molecules - Google Patents

Abstract

The present invention provides methods and compositions for performing high density molecular alignment and controlled spacing of nucleic acid molecules on a substrate. The aligned nucleic acid molecules can subsequently be subjected to genetic analysis.

Description

200912307 九、發明說明： 相關申請案之交互參照 本申請案主張2007年6月25日申請之美國臨時申請 案序號60/937,066之35 USC §119的權益，將其以引用^ 式納入本文中。 【發明所屬之技術領域】 本發明通常係關於為了生物分析，核酸分子在基質上 的排列’以及經控制之間隔。 【先前技術】 發明背景 存有在基質上以薄或單層進行多股核酸分子之低密度 分子排列的方法。某些方法集中在分離一或數股的材料= 並將其等拉開以便觀察並進行遺傳分析。這類方法的實例 為在美國專利第6,548,255號中使用之氣·水凹液面的分子 梳（m〇lecular c〇mbing)，以及如在美國專利第6，⑷，刚號 和美國專利申請案2005/0082204中描述之光學作圖的分子 排列技術。然而，對於進行多股核酸分子之高密度分子排 列的新穎方法仍存有需求。 【發明内容】 發明概述 為了在分析核酸分子（在一個或高達數個分子厚的一層 5 200912307 中）時，適用於其中希望判讀儀器有高生產率之處，將分子 更緊岔地一起排列，會改善過程的速度和經濟效益。具有 i控制間隔之分子的高密度排列，需要較少的判讀步驟， 因為在判讀儀視野中有較多的材I這在使用較高解析度 較P貝之判3賣系統來捕捉待分析目標物的更多細節時變 得曰益重要。在美國專利申請公開案編號美國專利第 7,291，468號、美國專利帛7,291，467 m、美國專利第 7’288,379號、美國專利申請案2〇〇6_〇〇24717和美國專利申 請案2007-0134699中描述了這類系統及其組件的實例，將 其全部以引用方式納入本文中。若利用加工維持或改善排 列之品質，同時仍允許區別該股，則該生產率和成本效率 是特別有價值的。 本發明一方面提供進行核酸分子之高密度分子排列的 方法。本發明一方面提供高密度間隔之核酸分子單層。在 某些具體事f令，㈣酸分子為多股的。本發日月包括在薄 膜基質上進行多股核酸分子之分子排列的方法，其結果產 生單層或部分單層的分子，在其等之間具有比其他方法明 顯更小的間隔。在某些具體事實中，該方法的第一個步驟 包括以預定之圖案或密度，在基質表面附接分子的一端。 在某些具體事實中，該方法的第二個步驟包括對該分子施 加一或排列力之組合（同時或連續地），使得其等直立並以想 要的方向排列。預定的附接圖案及空間密度技術（尤其是濃 度、溫度、pH值和鹽濃度控制）的使用，結果在施加排列力 (如流體流動）之後，分子便以緊密之間隔隔開。在某些具體 6 200912307 事實令，經排列之分子會與基質表面結合。在某些具體事 實中，修飾該基質表面，以容許經排列之分子結合。在某 些具體事實尹，第三個步驟包括將經排列之分子固定在表 面上’以維持高密度排列圖案。可在相同的基質上重複這 些步驟，以產生更複雜的圖案。 本發明一方面提供產製高密度間隔之核酸單層的方 法，該方法包括進行第一個附接步驟，以便將第一個核酸 分子族群之核酸分子的一端，附接到基質的第一群接觸點 上，其中該第一群接觸點係位在基質表面上的附接區中， 並使附接至第一群接觸點之核酸分子排列，產生第一組經 排列之核酸分子，其實質上是平行的，並以低於1微米之 間隔隔開’藉以使第一組經排列之核酸分子與基質表面結 合，藉此在基質的表面（其包括第一組經排列之核酸分子） 上產製而密度間隔之核酸分子單層。 在某些具體事實中，該方法更包括進行第二個附接# 驟’以便將第二個核酸分子族群附接到附接區的第二群指 觸點上，並使附接至第二群接觸點之核酸分子排列，以遽 生第二組經排列之核酸分子，其實質上是平行的，並&quot; 於J微米之間隔隔開，藉以使第二組經排列之核酸分子费 基質表面結合，藉此太其哲&amp;主 精此在基質的表面（其包括第一和第二組慈 排列之核酸分子)上產製高密度間隔之核酸分子單層。 ::文描述之方法的某些具體事實中，第一次附以 括、'由第一群連接子將第-個核酸分子族群與第一群㈣ 點附接。在某些具體事實中，第二次附接包括經由第二君 200912307 連接子將第一個核酸分子族群與第二群接觸點附接 些具體事實中，筮 v — 弟一個連接子是寡核苷酸。在某些具體事 實中，第一個連接子是寡核苷酸。在某些具體事實中，第 、個連接子包括正_辛基基團。在某些具體事實中，第二個 連接子包括正-辛基。 在本文描述之方法的某些具體事實中，第一或第二個 附接步驟包括藉著在基質表面與核酸分子之間形成一或多 f :固仏鍵’使核酸分子附接。在某些具體事實中，第一或 第個附接步驟包括uv_交聯、在麟酸基團和官能化表面之 間產生離子吸引力、使用經修飾之dna以結合其他的基質 表面修飾，及/或使用陽離子表面處理。 在本文描述之方法的某些具體事實中，排列包括指引 |體机動越過基質。在某些具體事實中，排列包括施加電 場於核酸分子上。在某些具體事實中，排列包括施加磁場 於核酸分子上。 , I本文描述之方法的某些具體事實中，基質包括排列 區。在某些具體事實中，排列致使核酸分子在基質之排列 區上排歹j在某些具體事實中，基質包括薄膜。在某些具 體事實中’ δ亥薄膜為奈米級的膜。在某些具體事實中，經 排列之核&amp;L刀子實質上是直線的。在某些具體事實中，基 質包括石墨表面。在某些具體事實中，經排列之分子是梯 狀構形。 在本文描述之方法的某些具體事實中，第一個核酸分 子族群是經標示之核酸分子。在某些具體事實中，第—個 8 200912307 核酸分子族群是未經標示之核酸分子。在某些具體事實 中，第二個核酸分子族群是經標示之核酸分子。在某些具 體事實巾，帛〔個核酸分子族群是未經標#之核酸分子。 在某些具體事實中’該標記是可使用穿透式電子：微鏡 (TEM)檢測的。在某些具體事實中，該標記包括具有核電荷 的原子，其可在得自TEM的影像上，U與背景雜訊對比來 檢測之。在某些具體事實中，該標記包括一至五個非螢光 原子。。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 TECHNICAL FIELD OF THE INVENTION The present invention generally relates to the arrangement of nucleic acid molecules on a substrate for biological analysis and the controlled spacing. [Prior Art] Background of the Invention There is a method of arranging low-density molecules of a plurality of nucleic acid molecules in a thin or single layer on a substrate. Some methods focus on separating one or several strands of material = and pulling them apart for observation and genetic analysis. An example of such a method is a molecular comb of gas/water recesses used in U.S. Patent No. 6,548,255, and U.S. Patent No. 6, (4), No. and U.S. Patent Application. The molecular alignment technique of optical mapping described in 2005/0082204. However, there remains a need for novel methods for performing high density molecular arrays of multiple strands of nucleic acid molecules. SUMMARY OF THE INVENTION Summary of the Invention In order to analyze nucleic acid molecules (in one layer or up to several molecular thicknesses of layer 5 200912307), where it is desired to have high productivity in the interpretation of the instrument, the molecules are arranged more closely together, Improve the speed and economics of the process. A high-density arrangement of molecules with i-controlled intervals requires fewer interpretation steps because there are more materials in the field of view of the interpreter than using the higher resolution than the P-selling system to capture the target to be analyzed. More details of the object become more important. U.S. Patent No. 7,291,468, U.S. Patent No. 7,291,467, U.S. Patent No. 7, 288, 379, U.S. Patent Application Serial No. PCT Application No. Examples of such systems and their components are described in U.S. Pat. This productivity and cost efficiency is particularly valuable if the processing is used to maintain or improve the quality of the arrangement while still allowing differentiation of the strand. One aspect of the invention provides a method of performing high density molecular alignment of nucleic acid molecules. One aspect of the invention provides a single layer of nucleic acid molecules of high density spacing. In some specific matters, (4) acid molecules are multi-stranded. The present day and the month include a method of performing molecular alignment of a plurality of nucleic acid molecules on a film substrate, which results in a single layer or a portion of a single layer of molecules having a significantly smaller spacing between them than other methods. In some specific instances, the first step of the method includes attaching one end of the molecule to the surface of the substrate in a predetermined pattern or density. In some specific cases, the second step of the method involves applying a combination of the elements or alignment forces (simultaneously or continuously) such that they are erected and aligned in the desired direction. The use of predetermined attachment patterns and spatial density techniques (especially concentration, temperature, pH, and salt concentration control) results in molecules being separated at close intervals after application of alignment forces (e.g., fluid flow). In some specific 6 200912307 facts, the aligned molecules bind to the surface of the substrate. In some specific instances, the surface of the substrate is modified to allow alignment of the aligned molecules. In some specific facts, the third step involves immobilizing the aligned molecules on the surface to maintain a high density pattern. These steps can be repeated on the same substrate to produce a more complex pattern. One aspect of the invention provides a method of producing a high density spacer nucleic acid monolayer, the method comprising performing a first attachment step to attach one end of a nucleic acid molecule of a first nucleic acid molecule population to a first population of substrates At a contact point, wherein the first group of contact points are ligated in an attachment region on the surface of the substrate, and the nucleic acid molecules attached to the first group of contact points are aligned to produce a first set of aligned nucleic acid molecules, the essence of which The upper layers are parallel and spaced at intervals of less than 1 micron to thereby bind the first set of aligned nucleic acid molecules to the surface of the substrate, thereby on the surface of the substrate (which includes the first set of aligned nucleic acid molecules) A single layer of nucleic acid molecules produced at a density of intervals. In some specific facts, the method further comprises performing a second attachment step to attach the second population of nucleic acid molecules to the second group of finger contacts of the attachment region and attaching to the second The nucleic acid molecules of the group of contacts are arranged to produce a second set of aligned nucleic acid molecules that are substantially parallel and are spaced apart by a J micron interval whereby the second set of aligned nucleic acid molecules are matrixed The surface is bound, whereby the genus &lt;RTIgt; </ RTI> </ RTI> </ RTI> <RTIgt; </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; In some specific facts of the method described in the text, for the first time, the first nucleic acid molecule population is attached to the first group (four) by the first group of linkers. In some specific cases, the second attachment includes the attachment of the first nucleic acid molecule population to the second group of contacts via the second Jun 200912307 linker, 筮v - the other linker is an oligo Glycosylate. In some specific cases, the first linker is an oligonucleotide. In some specific cases, the first linker includes a positive-octyl group. In some specific cases, the second linker includes a n-octyl group. In some specific aspects of the methods described herein, the first or second attachment step comprises attaching the nucleic acid molecule by forming one or more f: steric bonds between the surface of the substrate and the nucleic acid molecule. In certain specific instances, the first or first attachment step includes uv_crosslinking, ionic attraction between the linonic acid group and the functionalized surface, and the modified dna to bind other substrate surface modifications, And/or using a cationic surface treatment. In some specific instances of the methods described herein, the alignment includes directing the body to move over the substrate. In some specific instances, arranging includes applying an electric field to the nucleic acid molecule. In some specific instances, arranging includes applying a magnetic field to the nucleic acid molecule. In some specific facts of the method described herein, the matrix includes an alignment region. In some specific instances, the alignment causes the nucleic acid molecules to align on the aligned regions of the matrix. In some specific instances, the matrix comprises a thin film. In some specific facts, the film is a nano-scale film. In some specific cases, the aligned core &amp; L knives are substantially straight. In some specific cases, the matrix includes a graphite surface. In some specific cases, the aligned molecules are ladder-like configurations. In some specific instances of the methods described herein, the first population of nucleic acid molecules is the indicated nucleic acid molecule. In some specific cases, the first 8 200912307 nucleic acid molecule population is an unlabeled nucleic acid molecule. In some specific cases, the second population of nucleic acid molecules is the labeled nucleic acid molecule. In some specific facts, 帛[a group of nucleic acid molecules is a nucleic acid molecule that is not labeled #. In some specific facts, the mark can be detected using a transmissive electron: micromirror (TEM). In some specific cases, the mark includes an atom with a nuclear charge that can be detected on the image from the TEM, U in contrast to the background noise. In some specific cases, the label includes from one to five non-fluorescent atoms.

在本文描述之方法的某些具體事實中，該核酸分子的 長度為至少100個鹼基對。在某些具體事實中，該核酸分 子的長度為至少1000個鹼基對。在某些具體事實中，該核 酸分子的長度為至少10,000個鹼基對。在某些具體事實 中’該核酸分子的長度為至少2〇,〇〇〇個鹼基對。 在本文描述之方法的某些具體事實中，經排列之分子 與基質表面的結合是非-共價的。在某些具體事實中，經排 列之分子與基質表面的結合是可逆.的。在某些具體事實 中，與表面之結合包括使定向之流體流動減緩及/或停止、 改變流體及/或環境之溫度、在使流體流動停止後以定向之 方式使核酸分子脫水，及/或在移除流體之前或之後，加入 促進與表面之結合的試劑及/或活化劑。在某此且體事實 中，與表面之結合包括藉著在基質表面與核酸分子之間形 成一或多個共價鍵，使核酸分子附接。在某些具體事實中， 與表面之結合包括UV-交聯，在磷酸基團和官能化表面之間 產生離子吸引力’使用經修飾之DNA以結合其他的基質表 9 200912307 、t飾及/或使用陽離子表面處理。在某些具體事實中， 垂妾子# 4面修飾疋在基質上的相同區域。在某些具體事 只中〃表面之結合包括經由結合對使核酸分子附接至 面。 在本文描述之方法的某些具體事實中，至少一部分的 基質/面是疏水性的。纟某些具體事實中，至少一部分的 土貝表面疋、、、i g此化而成為疏水性的。在某些具體事實 中，同始度間隔之核酸分子單層為部分單層。 在本文描述之方法的某些具體事實中，至少25%經排 列之核酸分子以低於100奈米之間隔隔開。在某些具體事 貫中，至少50%經排列之核酸分子以低於丨〇〇奈米之間隔 隔開。在某些具體事實中，至少25%經排列之核酸分子以 低於10奈米之間隔隔開。在某些具體事實中，至少5〇%經 排列之核酸分子以低於1 〇奈米之間隔隔開。在某些具體事 實中’至少25%經排列之核酸分子是相碰觸的。在某些具 體事實申’至少50°/。經排列之核酸分子是相碰觸的。在某 些具體事貫中’至少2 5 /ί (經排列之核酸分子是以使經標示 和未經標示之核酸分子交替的方式來排列。在某些具體事 實中’至少50%經排列之核酸分子是以使經標示和未經標 示之核酸分子交替的方式來排列。 在本文描述之方法的某些具體事實中，使一或多個磁 珠附接於核酸分子上’並藉著施加磁力來排列該核酸分子。 本發明一方面提供高密度間隔之核酸分子單層，其包 括基質和經排列之核酸分子，後者以低於1微米之間隔隔 200912307 * 4並附接在基質上。在某些具體事實中 接在基質之第一群接觸點上的第— 4層包括附 此且體事實中，兮梦 t酸分子族群。在竿 一、體事實中’該第一群接觸點係位 在某 區中。在某些具體事實中，談單 、 上的附接 多個點上的第一组經排“匕括附接在基質表面之 J乐組經排列之核酸分 中，該單層包括附接在基質之第二群接觸體事實 酸分子族群。在某些且體 ，，上的第二個核 基質表面上的附接區中。 〜第二群接觸點係位在 在：文描述之單層的某些具體事實令，該基 列區。在某些具體事實令，該基質具有石墨表面。 ^本文描述之單層的某些具體事實中，經排列之核酸 :子包括第二組經排列之核酸分子，其附接在基質表面的 =固點上。在某些具體事實中’經排列之核酸分子實質上 疋直線的。在某些具體事實中’經排列之核酸分子為梯狀 構形。在某些具體事實令，經排列之核酸分子為經標示之 核酸分子。 西一在本文描述之單層的某些具體事實中，核酸分子為經 钛示和未經標示之核酸分子的混合物。在某些具體事實 中，該標記為可使用穿透式電子顯微鏡（TEM)檢測的。在某 些具體事實中’該標記包括具有可在得自TEM的影像上， 以與者景雜訊對比來檢測之分子量的原子。在某些具體事 貝中，戎標記包括一至五個非-螢光原子。 在本文描述之單層的某些具體事實中，該核酸分子的 長度為至少100個鹼基對。在某些具體事實中，該核酸分 200912307 · 子的長度為至少觸個驗基對。在某些具體事實中，該核 酸分子的長度為至少、10，_個鹼基對。在某些具體事實 中，該核酸分子的長度為至少20，_個鹼基對。在某些具 體事實中，該核酸分子藉著連接子附接在基f表面上。在 某些具體事實中’該核酸分子藉著在基質表面和核酸分子 之間的一或多個共價鍵，附接在基質表面上。 f 在本文描述之單層的某些具體事實中，至少一部分的 基質表面為疏水性的。纟某些具體事實巾，該纟質具有薄 膜。在某些具體事實中，該薄膜為奈米級的膜。在某些具 體事貝中至j一部分的基質表面是經官能化而成為疏水 性的。在某些具體事實中，該單層為部分單層。 在本文描述之單層的某些具體事實中’至少25%經排 狀核酸分子以低⑨ΠΚ)奈米之間隔隔開。在某些具體事 貫中，至少、50%經排列之核酸分子以低於1〇〇奈米之間隔 隔開。在某些具體事實中，至少25%經排列之核酸分子以 低於10奈米之間隔隔開。在某些具體事實中，至少經 排列之核酸分子以低於10奈米之間隔隔開。在某些具體事 實中，至少、25%經排列之核酸分子是相碰觸的=某些具 體事實中，m〇%經排列之核酸分子是相碰觸的。在某 些具體事實中，至少' 25%經排列之核酸分子是以使經標示 和未經標不之核酸分子交替的方式來排列。在某些具體事 實中，至少、50%經排列之核酸分子是以使經標經標 示之核酸分子交替的方式來排列。 在本文描述之單層的某些具體事實中，係藉著本文描 12 200912307 述之任何方法來生產該單層。 本發明一方面提供決定核酸族群之序列的方法，包括 獲得經標示之核酸分子族群，使用經標示之核酸分子族群 作為根據本文描述之任何方法的第第二個核酸分子族 群，產製高密度間隔之核酸分子單層，並使用粒子束判讀 单層之核酸分子的序列。 在某些具體事實中，該方法更包括藉著從試樣中獲得 核酸分子族群，產製經標示之核酸分子族群，並以一或多 個才示3己（其可使用穿透式電子顯微鏡（TEM)檢測）標示該核酸 分子之核發酸，以產製經標示之核酸分子族群。 本發明的每個限制均包含本發明的各種具體事實。因 此，預期本發明的每個限制均涉及可納入本發明每個觀點 内的任-元件或元件之組合。本發明在其應用丨，並不限 於在下列說明中陳述或在圖式中解釋的結構細節和組件配 置。本發明能夠以各種方式實行或進行其他的具體事實。 再者，在本文中使用的措詞和術語，《為了說明，而不應 將其視為限制。在本文中使用”包含”、，，包括，，、，，具有，，、” a有' 涉及及其變化，意指包括後文列舉的項目及其相 等物’以及額外的項目。 【實施方式】 較佳具體實例之說明 定義 除非在本文中另行定義，所有在本文中使用的技術和 13 200912307 科學名詞均具有與熟習本發明所屬之技藝人士普遍了解的 相同的意義。 “附接區”為在基質上經㈣的表面區，在那裡藉著共價 結合或-般技藝人士已知的其他技術，允許或引起分子與 基質表面結合。附接區是盆中為7!1/_1&amp; 士 疋八甲馮了附接連接子及/或核酸分 子而設置接觸點的區域。在某些具體事實中’附接區也是 其中核酸分子排列並形成高密度間隔之單層的區域。一基 質可具有一或多個附接區。 — 將’排列區”定義為基質中核酸分子排列並形成高密度 間隔之單層的區域。排列區之位置與附接區相鄰。排列區 可能有表面修飾，以允許經排列之分子與排列區結合。一 基質可具有一或多個排列區。 “薄膜”為基質具有較小厚度的區域。薄膜可以小於ι〇〇 奈求、小於90奈米、小於8〇奈米、小於7〇奈米、小於6〇 奈米、小於5〇奈米、小於40奈米、小於30奈米、小於2〇 奈米、小於H)奈米、小於8奈米、小於5奈米和小於3奈 广。在某些具體事實中，薄膜小於3G奈米。該厚度报適合 :排列分子的TEM(穿透式電子顯微鏡）分析。薄膜區且有厚 ^和材料之組合，其對粒子束之粒子束物種是充分透明 ，Μ允許在粒子束物種通過試樣和透明區之後，檢測粒 束物種與生物試樣在一或多個透明區之表面上的交互作 用。薄膜可以是排列區的一部分或附接區的一部分。在某 些具體事實中，該薄膜為奈米級的膜。 、 當在本文中定義時’，，接觸點，，為在基質上的特定點，可 14 200912307 視需要在基質之附接區，可將連接子或核酸分子附接於其 上。 將在分子排列後的，，高密度間隔”定義為在排列之後，在 分子之間的間隔小於1微米、小於500奈米、小於250奈 米、小於200奈米、小於15〇奈米、小於1〇〇奈米、小於 90奈米、小於80奈米、小於7〇奈米、小於6〇奈米、小於 50奈米、小於40奈米、小於3〇奈米、小於25奈米、小於 不米小於15奈米、小於10奈米、小於9奈米、小於 8奈米、小於7奈米、小於6奈米、小於5奈米、小於4奈 米、小於3奈米、小於2奈米、小於1奈米或0奈米。在 某二八體事實中，該分子是互相碰觸的。將碰觸定義為在 4寸定組合的條件下，包括考慮分子之幾何學和在分子之間 的排斥交互作帛’所能允許的分子彼此緊靠。在某些具體 事實中’ ?曰’隔是在下列的範圍β : a)在其等寬度h在分子 之間〇距離（即該分子是相接觸的），以及b)在其等寬度上， 在分子之間隔離為相鄰分子之平均寬度的十倍⑽。然而， ，所有具體事實中，間隔都會小w i微米。將長度定義為 二核糖酸鹽主鏈平行之分子的尺寸。將寬度定義為與核 磷酸鹽主鏈成直角且在局部基質表面之平面中的尺寸。 右刀子的平均寬度為大約二十二(22)埃，則高密度間 同曰是在範圍從零（〇)埃到兩百二十（22())埃之寬度尺寸上的 任何間隔。然而’本發明亦包含其他的寛度/長度比例，只 :門隔小⑨1微米即可。分子可以是任何長度或寬度，可 不同的材料製造，可具有不同的尺寸或其任何組合。 15 200912307 “ jA 了- ” , -序列依賴性分於序列依賴性的單股低聚物和非 其他形式的二Π:體藝事人實：，連糾 ηΜΔ八i 叙技藝人士已知的。在連接子和 丘Γ間的結合可以是可逆的或不可逆的、共價或非- 接子可包括-或多種類型的分子，並可具有任 何長度。纟某些具體事實中，第—群核酸使㈣—個連接 子，而第二群核酸使用第二個連接子。在某些具體事實中， 該連接子是與核酸分子之單股突出物互補的寡核苦酸。在 某些具體事實中，該寡核普酸與雙股亀序列結合。在某 些具體事實中，該連接子是疏水性分子。在某些具體事實 中，該連接子包括中鏈烴基團，如正_辛基基團。中鍵煙基 團能夠在適合的pH下結合dsDNA(參見，例如AUemand, J.-F·，Bensimon，D·等人1997. !^八的pH值依賴性之專一’ 結合和梳（pH-Dependent Specific Binding and c〇mbing 〇f DNA) Biophysical Journal. 2064-2070) 〇 將在基質上”經分子排列之”核酸分子定義為在基質上 的二（2)或多個核酸分子，在實質上平行或平行線上。高品 質的排列在任何點都沒有核酸分子的重疊，核酸分子&amp; $ 部平行的線上，且核酸分子沿著其全長實質上都是直的。 品質差強人意的分子排列，在普遍弄直且經排列之分子中 可包含分子的重疊，以及實質上平行但彎曲的線，或部分 區域的纏結。 核酸分子包括，但不限於脫氡核糖核酸（“DNA”）、核糖 核酸（“RNA”）、肽核酸（“PNA”）、其他的核酸聚合物類似物 16 200912307 和修飾，及其混合物。核酸分子可能是單股或多股的，或 單股和多股的混合物（例如具有為單股的部分和為多股的部 分）。多股的核酸分子包括，但不限於雙股dna、三股dna、 雙股RNA、嵌合的DNA/RNa雙股、DNA/pNA雙股和 RNA/PNA雙股。在國際專利公開案w〇2〇〇7/〇76i32令描述 了核酸的額外觀點。 如在本文中之定義，，，梯狀構形，，是其中兩個核糖_磷酸 鹽主鏈已經從DNA螺旋中鬆開，並已經變成直線且平行 的，就像由階梯連接之梯狀㈣個長邊，還有由沃森克里 克⑽tS〇n-Crick)驗基對形成階梯的構象。T藉著經由某種 分子排列方法控制螺旋的拉伸，使其遠離一附接點，而形 成梯狀構形。可藉著使職分子與表面結合，維持梯子形 狀。在梯狀構形中難驗基的組織化，允許改良核酸驗基 對的顯影。 單層是覆蓋基質的一層分子，其具有一個分子之厚 度。應明瞭在本發明中，，，單層，，意指覆蓋基質的單層和，，部 分早層，，兩者。部分單層，當在本文中使用時，為覆蓋基質 局P區域# |分子，對於該局部區域之實質部分而言， :有-個⑴分子的厚度’而在其他部分則沒有分子。在某 ::具體事實中’ 一部分的局部區域可具有超過一個⑴分子 苗：度。㊉了覆盍基質的局部區域之外’部分單層亦可覆 |其他分子薄層的局部區域。 中 基質是具有硬或半-硬表面的材料 基質的至少一個表面會是實質上平 。在許多具體事實 坦的，雖然在許多 17 200912307 具體事實φΑ , ，可能希望它具有流動通道及/或其他的機械排 ^ 如在基質上的拓撲學特徵，以促進分子排列。這 i道及/或其他機械排列輔助的實例包括’但不限於 挖溝可在其中部分或全部地放入一或多個分子， 、及在其間建立柵攔，可部份或全部地放入一或多個分 子。基質的尺寸範圍和材料為一般技藝人士已知的。 在某些具體事實中’基質具有附接區和排列區。在某 些具體事實中，基質沒有排列區。在某些具體事實中，附 接區和：列區含有不同的表面官能化。在某些具體事實 中基貝有一或多個薄膜和該薄膜的結構支撐物。 在某上具體事實中，本發明之方法有三個基本步驟： 吏杉i刀子端附接到基質上的步驟、使核酸分子排列的 步驟’以及在一或多個沿著分子之處(除了先前的附接端)， 使經排列之核酸分子與基質結合的步驟。最後一個步驟可 乂疋被動（主要經由非_共價結合）或主動的（進行反應以附 接經排列之分子至基質）。 可使用本發明之方法，在基質上以高密度間隔進行核 酸分子的分子排列。在某些具體事實中’ &amp; 了遺傳分析， 使用該方法在具有高密度間隔之dsDNA分子的部分單層上 進行分子排列。在某些具體事實中，已經標示讓分；， 以改善遺傳分析的品質。遺傳分析包括dsDNA分子的序列 匈定’以及經表現相的判定。遺傳分析亦包括&amp;崎的 定量判定和在試樣中以不同方式表現之核酸的判定。在本 文中將欲進行遺傳分析之DNA和核酸稱為，，分析物蘭a，,、,， 18 200912307 試樣DNA”、”欲分析之DNA”、”分析物核酸”、”試樣核酸” 和”欲分析之核酸”。 在某些具體事實中，使用該方法以便在具有高密度間 隔之dsDNA分子的部分單層上進行分子排列，其中一些核 酸分子有標記，其為具有核電荷（以原子序或’’Z”表示）的原 子，其可在得自穿透式電子顯微鏡（“TEM”）的影像上，以與 背景雜訊對比來檢測之，而一些核酸分子則沒有這類標 記。在某些具體事實中，經標示及/或未經標示之DNA分子 含有修飾，以支持排列並以間隔隔開。可使用該方法以進 行多股核酸分子的高密度分子排列，該多股核酸分子具有 超過但不限於：100、200、300、500、700、1000、1500、 2000、3000 ' 5000、7000、10000、15000、20000、25000、 30000、50000、70000、100000、200000 ' 500000、1000000 或更多個鹼基之長度。某些具體事實包括使用薄膜，之後 進行分子排列並以間隔隔開，接著是TEM分析。在某些具 體事實中，使用該方法以判定核酸分子（例如dsDNA)之序 列，判讀相鄰的5,000或更多，或10,000或更多，和20,000 或更多，或50,000或更多，或100,000或更多，或500,000 或更多，或1,000,000或更多個鹼基對。 以下的說明有時為了方便提到DNA或dsDNA分子，但 應將本發明所描述之具體事實解釋為可應用在任何多股的 核酸分子上。圖1為在欲描述之方法中三個步驟的計晝概 論。 將核酸分子的一端附接在基質上 19 200912307 本發明之方法的第一個步驟’是完成以經控制之圖 案’將多個.DNA分子的一端附接到基質上。選擇附接圖案， 結果產生DNA分子的高密度間隔測量值，其在排列之後， 提供或接近適合應用在遺傳分析上的想要或目標間隔。在 某些具體事實中，可與連接子附接的接觸點分散在整個附 接區中’且DNA分子可附接於整個基質。在某些具體事實 中’基質具有附接和排列區兩者，而DNA分子僅能附接在 基質區的子集上。 可藉著各種方法，獲得附接多個DNA分子一端的經控 制圖案。在一方法中，以預定的密度或圖案，利用連接子 裝備附接區。應明瞭連接子之間隔的密度不需與經排列 DNA分子之高密度間隔的最終密度是相同的，因為可在排 列步驟期間，藉著流動方向調整間隔密度（參見下文）。在某 些具體事實中，本發明之基質具有排列區，其具有預定的 連接子密度，而所得之排列區中的DNA分子具有高密度間 隔。 單獨或以組合方式，可決定經排列DNA分子之高密度 間隔的因f ’包括.1)連接子表面密度；2)附接區大小和形 狀，3)後續分子排列技術的方向。在段落和圖15中提供了 k二因素如何交互作用的實例。在所有的具體事實中，安 排以上的因素’結果產生經排列分子的高密度間隔。 在將連接子固定於在基質上的接觸點之後，在準備排 列步驟時，使DNA分子與連接子結合。附接連接子至基質 的方法為在技術領域中已知的，例如創造dna微陣列。結 20 200912307 合DNA分子與連接子的 … 于的方法亦為-般技藝人士已知的，並 包括但不限於直接丑僧级从 …… 與序列依賴性連接子雜交， 之連接子m補的其他鍵結。在某此具體 事實^料接子為寡料酸。欲㈣之亀分^長度 可能是多變的，且“ 又在其上發生排列之基質表面積的大 小限制。 一在某些具體事實t’在擴散和連接子與基質表面附接 之前，連接子和DNA分子業已在液體十結合。在某些具體 事實中’ S連接子和DNA之間有—或多個中間分子。在某 些具體事實中，DNA分子直接與基質接觸點結合，沒有分 開的連接+。先前描m)NA分+亦可能是單股的，並在 一端附接於基質之後，藉著PCR或其他方法將其轉變為多 股。 在某些具體事實中，必須移除無官能的連接子，以便 最優化經排列分子與基質表面的結合，.因為未反應之連接 子可能與核酸分子的排列產生交互作用。可藉著在進行附 接時增加核酸分子對連接子的比例，或藉著使未反應之連 接子驟冷或將其移除，使未反應之連接子的量減至最少。 DNA分子可以是經標示或未經標示的。標記之類型包 括，但不限於螢光分子、一至五個非-螢光原子，包括具有 高核電荷的原子，以及放射性分子。在國際專利公開案 W02007/076132和W02006/019903中描述了核酸標記的額 外觀點。In certain specific instances of the methods described herein, the nucleic acid molecule is at least 100 base pairs in length. In some specific instances, the nucleic acid molecule is at least 1000 base pairs in length. In some specific instances, the nucleic acid molecule is at least 10,000 base pairs in length. In certain specific instances, the nucleic acid molecule is at least 2 长度 in length, 〇〇〇 one base pair. In some specific instances of the methods described herein, the binding of the aligned molecules to the surface of the substrate is non-covalent. In some specific cases, the binding of the aligned molecules to the surface of the substrate is reversible. In some specific instances, the combination with the surface includes slowing and/or stopping the directed fluid flow, changing the temperature of the fluid and/or the environment, dehydrating the nucleic acid molecule in an oriented manner after stopping the fluid flow, and/or An agent and/or an activator that promotes binding to the surface is added before or after the fluid is removed. In some instances, binding to the surface includes attaching the nucleic acid molecule by forming one or more covalent bonds between the surface of the substrate and the nucleic acid molecule. In some specific cases, the combination with the surface includes UV-crosslinking, creating an ionic attraction between the phosphate group and the functionalized surface. Using modified DNA to bind other substrates. Table 9 200912307, t-and/or Or use a cationic surface treatment. In some specific cases, the scorpion #4 face is modified to the same area on the substrate. In some specific cases, only the binding of the surface comprises attaching the nucleic acid molecule to the surface via a binding pair. In some specific instances of the methods described herein, at least a portion of the substrate/face is hydrophobic.纟 In some specific facts, at least a part of the surface of the shellfish is 疏水, , i g is made hydrophobic. In some specific cases, a single layer of nucleic acid molecules of the same initial interval is a partial monolayer. In some specific instances of the methods described herein, at least 25% of the aligned nucleic acid molecules are separated by less than 100 nanometers. In some specific instances, at least 50% of the aligned nucleic acid molecules are separated by a spacing below the nanometer. In some specific instances, at least 25% of the aligned nucleic acid molecules are separated by less than 10 nanometers. In some specific instances, at least 5% of the aligned nucleic acid molecules are separated by an interval of less than 1 nanometer. In some specific instances, at least 25% of the aligned nucleic acid molecules are in contact. In some specific facts, at least 50°/. The aligned nucleic acid molecules are in contact with each other. In some specific cases 'at least 2 5 / ί (the aligned nucleic acid molecules are arranged in such a way that the labeled and unlabeled nucleic acid molecules alternate. In some specific facts, 'at least 50% are aligned Nucleic acid molecules are arranged in such a way as to alter the labeled and unlabeled nucleic acid molecules. In some specific instances of the methods described herein, one or more magnetic beads are attached to a nucleic acid molecule' and by application Magnetic field to align the nucleic acid molecules.In one aspect, the invention provides a high density interstitial nucleic acid molecule monolayer comprising a matrix and aligned nucleic acid molecules separated by a spacing of less than 1 micrometer 200912307*4 and attached to a substrate. In some specific facts, the fourth layer attached to the first group of contact points of the matrix includes the body composition of the nightmare t-acid molecule group. In the first, body facts, the first group of contact points Trapped in a certain area. In some specific facts, the first set of rows on a single point attached to a single sheet is included in the nucleic acid group of the J group that is attached to the surface of the substrate. The single layer includes attached to the substrate Two groups of contact body fact acid group of molecules. In some attachments on the surface of the second nuclear matrix, the second group of contact points are in the single layer of the description: The specific facts make the base region. In some specific facts, the matrix has a graphite surface. ^ In some specific facts of the single layer described herein, the aligned nucleic acids: the sub-region includes a second set of aligned nucleic acid molecules Attached to the = fixed point on the surface of the substrate. In some specific facts, the 'aligned nucleic acid molecules are substantially linear. In some specific facts, the aligned nucleic acid molecules are ladder-like. Certain specific facts indicate that the aligned nucleic acid molecules are labeled nucleic acid molecules. In certain specific facts of the single layer described herein, the nucleic acid molecule is a mixture of titanium-labeled and unlabeled nucleic acid molecules. In some specific cases, the mark can be detected using a transmission electron microscope (TEM). In some specific facts, the mark includes an image that can be obtained from the TEM to compare with the scene noise. Detected molecular weight Atom. In some specific cases, the ruthenium label includes one to five non-fluorescent atoms. In some specific facts of the single layer described herein, the nucleic acid molecule is at least 100 base pairs in length. In some specific facts, the length of the nucleic acid is at least one base pair. In some specific facts, the nucleic acid molecule is at least 10, _ base pairs in length. In some specific facts Wherein the nucleic acid molecule is at least 20, _ base pairs in length. In some specific instances, the nucleic acid molecule is attached to the surface of the base f by a linker. In some specific instances, the nucleic acid molecule One or more covalent bonds between the surface of the substrate and the nucleic acid molecule are attached to the surface of the substrate. f In some specific instances of the monolayer described herein, at least a portion of the surface of the substrate is hydrophobic. In some specific facts, the enamel has a film. In some specific cases, the film is a nanoscale film. The surface of the substrate in some of the specific things to the part j is functionalized to become hydrophobic. In some specific cases, the single layer is a partial single layer. In some specific instances of the monolayers described herein, &apos;at least 25% of the ligated nucleic acid molecules are separated by a low 9 Å. In some specific instances, at least 50% of the aligned nucleic acid molecules are separated by less than 1 nanometer. In some specific instances, at least 25% of the aligned nucleic acid molecules are separated by less than 10 nanometers. In some specific instances, at least the aligned nucleic acid molecules are separated by less than 10 nanometers. In some specific instances, at least 25% of the aligned nucleic acid molecules are in contact = in some specific facts, m〇% of the aligned nucleic acid molecules are in contact. In some specific instances, at least '25% of the aligned nucleic acid molecules are arranged in such a way as to alter the labeled and unlabeled nucleic acid molecules. In some specific instances, at least 50% of the aligned nucleic acid molecules are arranged in such a way that the labeled nucleic acid molecules are alternated. In some specific instances of the single layer described herein, the single layer is produced by any of the methods described herein. In one aspect, the invention provides a method of determining a sequence of a nucleic acid population comprising obtaining a population of labeled nucleic acid molecules, using a population of labeled nucleic acid molecules as a second population of nucleic acid molecules according to any of the methods described herein, producing a high density spacer The nucleic acid molecule is a single layer and the particle beam is used to interpret the sequence of the single layer nucleic acid molecule. In some specific cases, the method further comprises producing a population of labeled nucleic acid molecules by obtaining a population of nucleic acid molecules from the sample, and displaying one or more of them in one or more (which can use a transmission electron microscope) (TEM) detection) indicates the nuclear acid of the nucleic acid molecule to produce a population of labeled nucleic acid molecules. Each of the limitations of the invention encompasses various specific details of the invention. Therefore, it is intended that each of the limitations of the invention be construed as being in any combination of elements or elements that may be included in the various aspects of the invention. The present invention is not limited in its application, and is not limited to the details of the structure and the structure of The invention is capable of carrying out various other specific embodiments. Furthermore, the phraseology and terminology used herein is for the purpose of description and should not be construed as limiting. The use of "including", "includes", "includes", ",", ",", ",,,,,,,,,,,,,,,,, DESCRIPTION OF THE PREFERRED EMBODIMENT(S) Definitions Unless otherwise defined herein, all the techniques used herein and the scientific terminology of 13 200912307 have the same meaning as commonly understood by those skilled in the art to which the invention pertains. The surface region of (4) on the substrate, where it is allowed to bind or bind to the surface of the substrate by covalent bonding or other techniques known to those skilled in the art. The attachment zone is 7!1/_1&amp; A region in which a contact point is attached to a linker and/or a nucleic acid molecule. In some specific cases, the 'attachment region is also a region in which nucleic acid molecules are aligned and form a single layer of high-density spacers. Having one or more attachment zones - The 'arrangement zone' is defined as the region of the matrix in which the nucleic acid molecules are aligned and form a single layer of high density spacing. The position of the alignment area is adjacent to the attachment area. The alignment zone may have surface modifications to allow the aligned molecules to bind to the alignment regions. A substrate can have one or more alignment zones. A "film" is a region of a substrate having a small thickness. The film may be less than ι 〇〇, less than 90 nm, less than 8 〇 nanometer, less than 7 〇 nanometer, less than 6 〇 nanometer, less than 5 〇 nanometer, less than 40 nanometer, less than 30 nanometer, less than 2 〇 nanometer, less than H) nanometer, less than 8 nanometers, less than 5 nanometers and less than 3 nanometers. In some specific cases, the film is less than 3G nanometers. This thickness report is suitable for: TEM (transmissive electron microscopy) analysis of aligned molecules. The film region has a combination of thickness and material that is sufficiently transparent to the particle beam species of the particle beam, allowing the detection of the particle beam species and the biological sample one or more after the particle beam species pass through the sample and the transparent region Interaction on the surface of the transparent zone. The film may be part of the alignment zone or part of the attachment zone. In some specific cases, the film is a nanoscale film. As defined herein, the contact point, which is a specific point on the substrate, may be attached to the attachment region of the substrate, as desired, to which the linker or nucleic acid molecule can be attached. After the alignment of the molecules, the high-density interval is defined as the interval between the molecules after the alignment is less than 1 μm, less than 500 nm, less than 250 nm, less than 200 nm, less than 15 nm, less than 1 〇〇 nanometer, less than 90 nm, less than 80 nm, less than 7 〇 nanometer, less than 6 〇 nanometer, less than 50 nanometer, less than 40 nanometer, less than 3 〇 nanometer, less than 25 nanometer, less than Not less than 15 nm, less than 10 nm, less than 9 nm, less than 8 nm, less than 7 nm, less than 6 nm, less than 5 nm, less than 4 nm, less than 3 nm, less than 2 Nai Meter, less than 1 nm or 0 nm. In a certain fact, the molecules are in contact with each other. The touch is defined as a combination of 4 inches, including the geometry of the molecule and the molecule. The reciprocal interaction between the two allows the molecules to be close to each other. In some specific facts, the '曰' is in the following range β: a) at its equal width h, the distance between the molecules (ie The molecules are in contact with each other, and b) in their equal width, the average width of adjacent molecules separated between molecules Ten times (10). However, in all specific cases, the spacing will be small wi microns. The length is defined as the size of the molecule in which the dinucleotate backbone is parallel. The width is defined as being at right angles to the core phosphate backbone and locally The dimension in the plane of the surface of the substrate. The average width of the right knife is about twenty-two (22) angstroms, and the high density is the same range from zero (〇) angstrom to two hundred twenty (22 ()) angstroms. Any spacing in the width dimension. However, the present invention also includes other twist/length ratios, only: the door spacing is 91 microns. The molecules can be of any length or width, can be made of different materials, and can have different sizes. Or any combination thereof. 15 200912307 "jA--", - sequence-dependent single-strand oligomers in sequence-dependent and non-other forms of diterpenes: physical arts: real, 纠ηΜΔ八i It is known that the binding between a linker and a scorpion can be reversible or irreversible, a covalent or non-linker can comprise - or a plurality of types of molecules, and can have any length. In some specific facts , the first group of nucleic acid makes (four) - a linker, and the second group of nucleic acids uses a second linker. In some specific cases, the linker is an oligonucleotide that is complementary to a single protrusion of a nucleic acid molecule. In some specific cases, The oligonucleotides bind to the double-stranded ruthenium sequence. In some specific cases, the linker is a hydrophobic molecule. In some specific instances, the linker comprises a medium chain hydrocarbon group, such as a n-octyl group. The mid-chain tobacco group is capable of binding dsDNA at a suitable pH (see, for example, AUemand, J.-F., Bensimon, D. et al. 1997. !^8, pH-dependent specificity' binding and combing (pH- Dependent Specific Binding and c〇mbing 〇f DNA) Biophysical Journal. 2064-2070) 核酸 The "molecularly aligned" nucleic acid molecule on a substrate is defined as two (2) or more nucleic acid molecules on a substrate, in essence Parallel or parallel lines. The high quality arrangement has no overlap of nucleic acid molecules at any point, the nucleic acid molecules &amp; $ parallel lines, and the nucleic acid molecules are substantially straight along their entire length. Molecular arrangements of poor quality can include overlapping molecules in a generally straightened and aligned molecule, as well as substantially parallel but curved lines, or entanglements of partial regions. Nucleic acid molecules include, but are not limited to, deuterated ribonucleic acid ("DNA"), ribonucleic acid ("RNA"), peptide nucleic acid ("PNA"), other nucleic acid polymer analogs 16 200912307 and modifications, and mixtures thereof. Nucleic acid molecules may be single or multi-stranded, or a mixture of single and multiple strands (e.g., having a single-stranded portion and a multi-strand portion). Multiple strands of nucleic acid molecules include, but are not limited to, double-stranded DNA, three-stranded DNA, double-stranded RNA, chimeric DNA/RNa double-stranded, DNA/pNA double-stranded, and RNA/PNA double-stranded. Additional views of nucleic acids are described in International Patent Publication No. 2〇〇7/〇76i32. As defined herein, the ladder configuration, where two ribose-phosphate backbones have been loosened from the DNA helix, and has become straight and parallel, like a ladder connected by a step (4) On the long side, there is a conformation formed by Watson Creek (10) tS〇n-Crick). T forms a ladder-like configuration by controlling the stretching of the helix by a molecular alignment method away from an attachment point. The shape of the ladder can be maintained by combining the incumbent with the surface. The organization of the hard-to-detect base in the ladder configuration allows for improved development of the nucleic acid test. A single layer is a layer of molecules covering the matrix that has a molecular thickness. It should be understood that in the present invention, a single layer means a single layer covering the substrate and, a portion of the early layer, both. A portion of the monolayer, as used herein, covers the matrix P region # | molecule, for a substantial portion of the local region: there is - (1) the thickness of the molecule 'and no other molecules in the other portion. In a :: specific fact, a portion of a localized region may have more than one (1) molecular seedling: degree. Ten parts of the outer layer of the substrate are covered. Partial single layer can also cover the local area of other thin layers of molecules. The medium substrate is a material having a hard or semi-hard surface. At least one surface of the substrate may be substantially flat. In many specific facts, although in many 17 200912307 the concrete facts φΑ, it may be desirable to have flow channels and/or other mechanical rows such as topological features on the substrate to facilitate molecular alignment. Examples of such i-channel and/or other mechanical alignment aids include, but are not limited to, trenching in which one or more molecules may be placed in part or in whole, and a barrier is established therebetween, which may be partially or fully placed One or more molecules. The size range and materials of the matrix are known to those of ordinary skill in the art. In some specific instances, the matrix has an attachment zone and an alignment zone. In some specific cases, the matrix has no alignment zones. In some specific cases, the attachment zone and the column zone contain different surface functionalizations. In some specific instances, the base has one or more films and structural supports for the film. In a particular fact, the method of the invention has three basic steps: a step of attaching the knives of the cedar i to the substrate, a step of arranging the nucleic acid molecules, and one or more along the molecule (except prior to Attachment), the step of binding the aligned nucleic acid molecules to the substrate. The last step can be either passive (primarily via non-covalent bonding) or active (reacting to attach the aligned molecules to the matrix). The molecular arrangement of the nucleic acid molecules can be carried out at high density intervals on the substrate using the method of the present invention. In some specific cases, &lt;&gt; genetic analysis, using this method to perform molecular alignment on a partial monolayer of dsDNA molecules with high density spacing. In some specific facts, a handicap has been marked; to improve the quality of the genetic analysis. Genetic analysis includes the sequence of the dsDNA molecule Hungarian' and the judgment of the expressed phase. Genetic analysis also includes the quantitative determination of &amp; Saki and the determination of nucleic acids that are expressed differently in the sample. The DNA and nucleic acid to be subjected to genetic analysis are referred to herein as, the analytes a,,,, 18 200912307 sample DNA", "DNA to be analyzed", "analyte nucleic acid", "sample nucleic acid" And "nucleic acid to be analyzed." In some specific cases, the method is used to perform molecular alignment on a partial monolayer of dsDNA molecules having high density intervals, some of which have a label, which has a nuclear charge (in An atomic sequence or an atom of ''Z') that can be detected on a image obtained from a transmission electron microscope ("TEM") in contrast to background noise, while some nucleic acid molecules do not have such a label. . In some specific cases, labeled and/or unlabeled DNA molecules contain modifications to support alignment and are separated by intervals. The method can be used to perform high density molecular alignment of a plurality of nucleic acid molecules having more than, but not limited to: 100, 200, 300, 500, 700, 1000, 1500, 2000, 3000 '5000, 7000, 10000 , 15000, 20000, 25000, 30000, 50000, 7000, 100000, 200000 '500000, 1000000 or more bases in length. Some specific facts include the use of a film followed by molecular alignment and separation by spacing, followed by TEM analysis. In some specific cases, the method is used to determine the sequence of a nucleic acid molecule (eg, dsDNA), contiguous 5,000 or more, or 10,000 or more, and 20,000 or more, or 50,000 or more, or 100,000 Or more, or 500,000 or more, or 1,000,000 or more base pairs. The following description sometimes refers to DNA or dsDNA molecules for convenience, but the specific facts described herein should be construed as being applicable to any multi-stranded nucleic acid molecule. Figure 1 is an overview of the three steps in the method to be described. Attaching one end of the nucleic acid molecule to the substrate 19 200912307 The first step of the method of the invention is to complete attaching one end of a plurality of DNA molecules to the substrate in a controlled pattern. The attachment pattern is selected, resulting in a high density interval measurement of the DNA molecule that, after alignment, provides or approximates the desired or target interval suitable for use in genetic analysis. In some specific cases, the contact points that can be attached to the linker are dispersed throughout the attachment zone&apos; and the DNA molecules can be attached to the entire matrix. In some specific instances, the matrix has both attachment and alignment regions, and DNA molecules can only be attached to a subset of the matrix regions. A controlled pattern to which one end of a plurality of DNA molecules is attached can be obtained by various methods. In one method, the attachment zone is equipped with a linker at a predetermined density or pattern. It should be understood that the density of the spacer spacers need not be the same as the final density of the high density spacing of the aligned DNA molecules, since the spacer density can be adjusted by the flow direction during the alignment step (see below). In some specific instances, the matrix of the present invention has an alignment region having a predetermined linker density, and the resulting DNA molecules in the alignment region have a high density interval. Alternatively or in combination, the high density spacing of the aligned DNA molecules can be determined by f&apos; including .1) linker surface density; 2) attachment zone size and shape, and 3) the direction of subsequent molecular alignment techniques. An example of how the k-factor interacts is provided in the paragraphs and in Figure 15. In all of the specific facts, the above factors are arranged to produce a high density interval of aligned molecules. After the linker is immobilized to the contact point on the substrate, the DNA molecule is bound to the linker at the time of preparation of the alignment step. Methods of attaching a linker to a substrate are known in the art, such as creating a dna microarray. Knot 20 200912307 DNA molecules and linkers are also known to those skilled in the art and include, but are not limited to, direct ugly levels from ... hybridization with sequence-dependent linkers, the linker m complements Other key knots. In some specific facts, the material is oligoacid. The length of the desired (4) may be variable, and "the size of the surface area of the substrate on which the alignment occurs is limited. One is in some specific facts t' before the diffusion and attachment of the linker to the surface of the substrate, the linker And DNA molecules have been combined in liquid ten. In some specific facts there are - or multiple intermediate molecules between the S linker and the DNA. In some specific facts, the DNA molecule binds directly to the substrate contact point, without separate Connection +. Previously m) NA score + may also be single-stranded, and after being attached to the substrate at one end, it is converted into multiple strands by PCR or other methods. In some specific facts, it must be removed. a functional linker to optimize binding of the aligned molecules to the surface of the substrate, since unreacted linkers may interact with the arrangement of the nucleic acid molecules. The ratio of nucleic acid molecules to linkers can be increased by attachment. , or by quenching or removing unreacted linkers, to minimize the amount of unreacted linkers. DNA molecules can be labeled or unlabeled. Types of tags include, but not It is limited to fluorescent molecules, one to five non-fluorescent atoms, including atoms having a high nuclear charge, and radioactive molecules. An additional view of nucleic acid labeling is described in International Patent Publications WO2007/076132 and WO2006/019903.

在 具體事實中，將欲附接至表面的分子（連接子、DN A 21 200912307 分子或一組合’可視需要包含中間分子）與具有類似尺寸的 其他分子（即所謂的”填料分子，，或，，嚮導分子”）混合。在某些 具體事實中，嚮導分子的尺寸比欲分析之DNA分子大。較 佳的是將該分子之混合納入流體或其他介質中，以使得混 合更容易。這些嚮導分子在表面上佔用了預定量的空間， 基於其等在核酸分子之混合物中的比例，因此打算為了附 接而將核酸分子稀釋。 應明瞭亦可藉著先將嚮導分子附接到連接子上，並在 後續的步驟中將欲分析之DNA分子附接到連接子上，製備 包括欲分析之DNA分子和嚮導分子兩者的高密度間隔。在 某些具體事實中，與第一個分子（例如嚮導分子）的接觸，散 佈在第二個分子（例如欲分析之DNA分子）的接觸點之間。 在某些具體事實中，按預定圖案排列第一個分子的連接子 和第—個分子的連接子，以產生具有預定圖案的排列。 在某些具體事實中，在附接嚮導分子之前，先附接欲 刀析之分子。在某些具體事實中，以不同種類的連接子㈠列 如具有不同序列的养核苷酸）附接嚮導分子和欲分析之分 子在某些具體事實中，排列嚮導分子以產生排列通道， 可在其中排列欲分析之分子。 在分子附接和嚮導分子的排列之後，以預定方式排列 連接子或其他分子，成為欲分析之DNA分子（亦稱為分析物 :子)的附接點。嚮導分子亦可併入校準輔助物，如定期標 或特殊序列，其拐不（例如藉著推論）核酸分 他特性，或得自試樣之相„記。 ^ 22 200912307 在某些具體事實中’使用包括但不限 或太半幼;'、子力顯微鏡 a不未_、，及千版印刷程序的技術， 卜且女π — η + 牧卞附接到在附接區 八有預疋®㈣接觸點上。在某些具體事實中 ===區時覆蓋排列區，或藉著對附接區和排 的，將連接其中該排列區對連接子是無反應性 J 將連接子限制在附接區。 〜在該方法之第一個步驟的先前說明中，陳述三個決定 =度間隔的因素；1)連接子表面密度，2)連接子所在之附 妾[大小和形狀，以及3)後續分子排列的方向。這 下文的數個實例來解釋。 曰# ▲第一個實例說明排列的方向如何與附接區尺寸和形狀 :互：用而影響高密度間隔。圖2“2b解釋本實例，並 =子排列，方向如何影響特定（不對稱）附接區的高密 又間隔。在本實例中，附接p·為_ ^&amp; 附接£為二（3)奈米（“nm”）乘七（7)奈 =的長方形’而連接子表面密度結果在表面的附接區中附 接了三（3)個連接子，各具有—⑴奈米之直徑。絲連接子 _在附接區’隨機表面結合的結果會使連接子在七⑺夺 米長度中形成相隔一⑴奈米，排成-直線的三(3)個分子， ^在三（3)奈米寬度中，使每個分子與邊之間相隔一⑴奈 未。然後將長雇股（例如一千〇,〇〇〇)奈米長）附接到連接 子。若後續的排列（以任一方向）通過長方形附接區的七⑺ 奈米邊（即以該邊的方向流動），則高密度間隔會是大約-⑴ 奈未。若後續的排列通過長方形附接區的三(3)奈米邊，則 南密度間隔通常會是零（〇)奈米，因為上游的腿分子（排 23 200912307 列流體流過來的方向）會流到下 此鄰接。 子的那—邊，並保持彼 /在圖3中出示第二個實例，其解釋附接區之尺寸和不 對稱的形狀如何與分子排列 間隔。m 交互作用而影響高密度 接子分子的表面密度與在第一個實例中 但附接區形狀為六（6)夺米乘 ' ° 八彼此上 ）不卡豕七（7)奈米。作為例證，連接子 冒擴散成大約二（2)行的三（3)個 兩⑺個並排的在第一個實例)中之 = 右「限制在附接區），像 -ί-π ^ 之附接區。然後將長度為一 、^方 之嶋股附接到連接子上。若後續的排列通 許全部六⑹個之間的高密丄=許四⑷個分子和或 有新的上游魏分子；;度在\=常、會是零⑼奈米，因為 個實例中隔開的空間在職分子之間，如同在第一In a specific case, the molecule to be attached to the surface (linker, DN A 21 200912307 molecule or a combination 'optionally containing intermediate molecules) and other molecules of similar size (so-called "filler molecules", or, , the guide molecule ") mixed. In some specific cases, the size of the guide molecule is larger than the DNA molecule to be analyzed. Preferably, the mixing of the molecules is incorporated into a fluid or other medium to make mixing easier. These guide molecules occupy a predetermined amount of space on the surface, based on their proportion in the mixture of nucleic acid molecules, and are therefore intended to dilute the nucleic acid molecules for attachment. It should be understood that by attaching the guide molecule to the linker first, and attaching the DNA molecule to be analyzed to the linker in a subsequent step, the preparation of the DNA molecule and the guide molecule to be analyzed is high. Density interval. In some specific cases, contact with the first molecule (e. g., a guide molecule) is interspersed between the contact points of the second molecule (e.g., the DNA molecule to be analyzed). In some specific cases, the linker of the first molecule and the linker of the first molecule are arranged in a predetermined pattern to produce an arrangement having a predetermined pattern. In some specific cases, the molecules to be analyzed are attached before the guide molecules are attached. In some specific cases, different types of linkers (a), such as nucleotides with different sequences, are attached to the guide molecule and the molecule to be analyzed. In some specific facts, the guide molecules are arranged to produce an alignment channel. The molecules to be analyzed are arranged therein. After the molecular attachment and alignment of the guide molecules, the linker or other molecule is arranged in a predetermined manner to become the attachment point of the DNA molecule (also referred to as the analyte: sub) to be analyzed. The guide molecule can also incorporate calibration aids, such as periodic or special sequences, which do not (eg, by inference) the nucleic acid's characteristics, or the phase of the sample. ^ 22 200912307 In some specific facts 'Use includes but not limited to or too young; ', sub-microscope a not _,, and the technology of the thousand-page printing program, Bu and female π - η + 卞 卞 attached to the attachment area ® (4) on the contact point. In some specific facts, the === area covers the alignment area, or by the attachment area and the row, which will be connected, the alignment area is not reactive to the linker J. In the attachment area. ~ In the previous description of the first step of the method, three factors determining the degree of interval are stated; 1) the density of the connected subsurface, 2) the attachment of the linker [size and shape, and 3) The direction of subsequent molecular alignment. This is explained by several examples below. 曰# ▲ The first example shows how the orientation of the alignment differs from the size and shape of the attachment area: mutual use affects the high-density interval. Figure 2 “2b Explain this example, and = subarray, how the direction affects the specific (asymmetry The high density and spacing of the attachment area. In this example, the attachment p· is _ ^&amp; attaches a rectangle of two (3) nanometers ("nm") by seven (7) nai = and the attachment surface density results on the surface of the attachment Three (3) connectors are attached to the zone, each having a diameter of - (1) nanometer. The results of the silk-linker _ in the attachment zone's random surface binding result in a linker forming one (1) nanometer apart in a seven (7) length of rice, three (3) molecules arranged in a straight line, ^ in three (3) In the width of the nanometer, each molecule is separated from the sides by one (1). Then attach the long-term shares (for example, one thousand 〇, 〇〇〇) to the linker. If the subsequent alignment (in either direction) passes through the seven (7) nanoside of the rectangular attachment zone (ie, flows in the direction of the edge), the high density interval will be approximately -(1) Nai. If the subsequent alignment passes through the three (3) nanometer sides of the rectangular attachment zone, the south density interval will usually be zero (〇) nanometers because the upstream leg molecules (the direction in which the fluid flows from the row 23 200912307) will flow. To the next contiguous. The side of the child, and the other, is shown in Fig. 3, which explains how the size of the attachment zone and the shape of the asymmetry are spaced from the molecular arrangement. The interaction of m affects the surface density of high-density linker molecules in the first instance but the shape of the attachment zone is six (6) and the number of meters is multiplied by '° eight on each other) not 豕 seven (7) nm. By way of example, the linker diffuses into approximately two (2) rows of three (3) two (7) side by side in the first instance) = right "limited in the attachment area", like -ί-π ^ Attachment area. Then attach the length of the 嶋 strand of the square to the linker. If the subsequent arrangement allows all six (6) high-density 许 = Xu (4) molecules and or new upstream Wei Molecules; degrees in \= often, will be zero (9) nanometers, because the space separated by an instance is between the incumbent molecules, as in the first

:三個實例顯示在液體中不同密度的排列分子如何與 附接區尺寸和飛此# A: Three examples show how different arrays of molecules in a liquid are attached to the size of the attachment area and fly this #A

互作用而影響高密度間隔。圖4A和4B 響。本實例使用在整個基質上維持不變的連接子 之表面密度。在三（3)奈米乘三（3)奈米的附接區_有 于右連接子的表面密度變成三倍，此時在相同大 I:::區”有大約二十七(27)個分子。這會在排列之後 二二:度、間隔’因為對於相同的直線寬度(在定向排列期 曰 /刀子會移動以安放於其t)，會有三倍之多的上游 魏分子（在與連接子結合之後）。 夕的上游 稭著使用本發明之方法，可控制並選擇核醆分子的排 列’使得核酸分子相隔小於！微米。使用在本文中描述之 24 200912307 方法意外地達成該接近，而得以獲得經排列核酸分孑之意 外密集的包裝。更特定而言，在某些具體事實中，在單層 中至少有2 5 %經排列之核酸分子以小於}微米之問隔隔 開，較佳的是相隔小於大約：900奈米、800奈来、7〇0奈 米、600奈米、5〇〇奈米、4〇〇奈米、300奈米、2〇〇奈米、 100奈米、90奈米、80奈米、7〇奈米、6〇奈米、5〇奈米、 40奈米、30奈米、20奈米、1〇奈米、9奈米、8奈米、7 奈米、6奈米、5奈米、4奈米、3奈米、2奈米、1奈米、 0.9奈米、0.8奈米、〇.7奈米、〇·6奈米、〇 5奈米、〇 4奈 米、0.3奈米、〇.2奈米或〇1奈米。在某些具體事實中，該 分子相隔2.5奈米到3.5奈米之間。在某些具體事實中，經 排列之核酸是相鄰的。在某些具體事實中，經排列之核酸 在超過其長度之至少大約：10%、2〇%、3〇%、4〇%、5〇0/〇、 60%、70%、80%、90%或95%是相碰觸的。較佳的是至少 大約· 10〇/〇、20%、30%、40%、50%、60%、70%、80%、 9 0 /。或9 5 %的經排列核酸分子以小於上文提及之距離相隔 (或彼此相碰觸）。 應明瞭在以上實例中之接觸點和連接子的精確排序僅 為了獲得具有不同間隔之高間隔密度而舉例說明，且可使 用接觸點、流動方向及其他參數的各種構型，以獲得本發 明之高密度間隔。 如在本文中提及的，在某些具體事實中，在未經標示 之核酸（例如嚮導分子）的存在下，排列經標示之核酸。在其 中排列經標示和未經標示之核酸分子兩者的具體事實中， 25 200912307 較佳的是以使經標示和未經標示之核酸分子交替的方式， 排列經排列之核酸分子’例如，如在圖3和圖5中所示。 較佳的是以交替方式排列至少大約：1〇%、2〇%、3〇%、4〇0/〇、 5 0%、60%、70%、80%、90。/❶、95%或 100%的經標示和未 經標不之核酸分子。 其他較佳的具體事實為與緊接在前之具體事實相同 的’其額外地使用經標示和未經標示之DNA分子的預定混 合物，後者為嚮導分子❶經標示之DNA分子典型地，但不 定是得自患者或欲分析之目標試樣的DNA。經標示之 DNA分子的標記可以是一般技藝人士已知的任何類型，包 括但不限於螢光標記和放射性標記。標記亦可由一到五個 明顯比平均DNA分子量更重的原子構成，如在經公開之專 利申清案美國專利第7,291,468號、美國專利第7,291，467 號美國專利第7,288,379號和W02006/019903中進一步描 述的，關於標記的相關教示，全部以引用方式納入本文中。 在某些具體事實中，嚮導分子是未經標示的DNA分 子，較佳的是其並非得自患者或目標試樣，但應該具有類 似絰如不DNA分子的物理特性（長度、寬度、密度及/或其 他特性）。嚮導分子可以有或可以沒有與經標示之分子結合 的犯力，例如藉著序列專一的或非-序列專一的結合。 使用未經標示之核酸分子，提供了另一種控制高密度 隔的方法。將未經標示之核酸分子與經標示之核酸分子 通機&amp; 5。隨著在混合物中未經標示核酸分子之百分比的 增加，經標示之核酸分子平均而言會越來越分散（一旦附接 26 200912307 在表面上）’亚降低兩個經標示之核酸分子會相鄰及/或碰觸 的可此性。忒控制是有價值的，因為經標示之核酸分子有 最小的距離’對於增加判讀系統對標記之效力可能是較佳 的。例如’若兩個經標示之分子彼此相鄰，則—個核酸分 子的軲η己可不易或無法與在另一個核酸分子上的標記區 別。此外，多股核酸分子自我-組織的天然特性，當核酸分 子彼此完全相鄰時’會在分子排列中創造較大的均—性。 利用該經標示和未經標示之核酸分子的具體事實，即使在 將表面上附接點的密度靶定在幾乎是零高密度間隔，並因 此達到高均-性時，仍可達成經標示之核酸分子的分離。 雖然通常會在與未經標示之核酸分子混合之前，先桿示試 樣核酸分子’但可在該方法之過程中的任何時間點 在某些具體事實中，在本發明方法的第一回合中，在 基質區内附接並排列嚮導分子。隨後，進行第二回合，在 相同的基質區中圍著嚮導分子附接並排列分析物分；。嚮 導分子可以是相當長的，例如在職雙股中有數十萬個驗 基對，以使分子排列步驟的效力和均—性達到最大。沿著 嚮導分子的全長，以防止其等在第 /〇 W 期間因分析物分 =排列力而脫離的方式附接該嚮導分子。通常，不標示 嚮導分子，以避免在排列後分析的期㈤，與經標亍之八析 物分子混淆 '然而’可利用與分析物分子之標記不同：標 δ己來裝備嚮導分子，以允許嚮導的定 此亚糟此定位分析 物分子。嚮導分子可作為在基質表面 工87相弓丨或通道邊 27 200912307 緣，以改良分析物分子的排列（亦參見圖13)。 至於具有薄膜區的基質，附接可發生在膜區或附近， 但排列和間隔控制實質上應該是在基質的膜區。換句話 說，附接區可包含薄膜區和基質的另一相鄰區兩者。 在某些具體事實中，基質僅具有附接區。在某些具體 事實中’基質具有附接區和排列區兩者。在圖12中舉例說 明僅有附接區的基質。在該具體事實中，將每個分析物分 子的一端附接在基質上會進行附接和排列兩者的區域。基 質區的大小、分析物分子的長度和核酸分子之目標間隔， 決定了在正與基質接觸之液體中核酸分子的量。分子越 長’將排列力下游之分子重疊減至最少所需的目標間隔就 越寬。步驟#ι，按照前述進行分子之—端及/或連接子的經 控制附接。步驟#2和#3，亦按照前述進行完整分子的分子 排列和附接。可將核酸附接在基質上的任何區域。在某些 具體事實中，連接子或接觸點的數目限制了在附接反應期 間所加入之核酸分子的量。限制未反應之連接子的量是適 當的，因為未反應的連接子分子仍可用來結合經排列之分 子，並干擾排列反應。在某些具體事實中，附接區包括連 接子（用於附接核酸的末端）和基質修飾（用以結合經排列之 核酸）兩者。 在圖2中舉例說明有附接和排列區兩者的基質，其中 以’’排列方向，，箭號表示排列區。可隨著流動方向的改變，改 變經排列分子的密度（亦參見下文）。 可使用在技 基質可能是全部或部分官能化的。例如 28 200912307 術領域中已知的技術和試劑，將基質的表面官能化以產生 疏水性表面（參見例如Allemand，Bensim〇n，等人，“DNA的 PH值-依賴性之專一結合和梳Interaction affects high density intervals. Figures 4A and 4B ring. This example uses the surface density of the linker that remains constant throughout the substrate. In the attachment area of three (3) nanometers by three (3) nanometers, the surface density of the right linker becomes three times, at this time in the same large I::: zone" there are about twenty-seven (27) Molecule. This will be after the arrangement of two or two degrees, interval 'because for the same line width (in the orientation period / knife will move to place it in t), there will be three times as many upstream Wei molecules (in connection with After the sub-combination), the upstream of the stalk can be used to control and select the arrangement of the ruthenium molecules 'so that the nucleic acid molecules are separated by less than ! microns. This approach is unexpectedly achieved using the method of 2009 2009307, described herein. Obtaining an unexpectedly dense package of aligned nucleic acid bifurcations. More specifically, in some specific cases, at least 25% of the aligned nucleic acid molecules in a single layer are separated by less than a micron. The best is less than about: 900 nm, 800 Nai, 7 〇 0 nm, 600 nm, 5 〇〇 nano, 4 〇〇 nano, 300 nm, 2 〇〇 nano, 100 nm , 90 nm, 80 nm, 7 〇 nano, 6 〇 nano, 5 〇 nano, 40 nm, 30 nm, 20 nm, 1 〇 nano, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, 1 nm, 0.9 Nano, 0.8 nm, 〇.7 nm, 〇·6 nm, 〇5 nm, 〇4 nm, 0.3 nm, 〇.2 nm or 〇1 nm. In some specific facts The molecules are separated by between 2.5 nm and 3.5 nm. In some specific cases, the aligned nucleic acids are contiguous. In some specific instances, the aligned nucleic acids are at least about 10: %, 2〇%, 3〇%, 4〇%, 5〇0/〇, 60%, 70%, 80%, 90% or 95% are in contact. Preferably at least about 10〇/ 〇, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 915% of aligned nucleic acid molecules are separated by less than the distances mentioned above (or touch each other) It should be understood that the precise ordering of the contact points and the linkers in the above examples is exemplified only to obtain high separation densities with different intervals, and various configurations of contact points, flow directions and other parameters can be used to obtain High density spacing of the present invention. As in this document Mentioned, in certain specific instances, the arrangement of labeled nucleic acids in the presence of unlabeled nucleic acids (eg, guide molecules), in the specific facts in which both labeled and unlabeled nucleic acid molecules are arranged 25 200912307 Preferably, the aligned nucleic acid molecules are arranged in such a manner that the labeled and unlabeled nucleic acid molecules are alternated, for example, as shown in Figures 3 and 5. Preferably, they are arranged in an alternating manner. At least approximately: 1〇%, 2〇%, 3〇%, 4〇0/〇, 50%, 60%, 70%, 80%, 90%/❶, 95% or 100% of the marked and not A nucleic acid molecule that is not labeled. Other preferred specific facts are the same as the exact facts immediately preceding it, which additionally uses a predetermined mixture of labeled and unlabeled DNA molecules, the latter being a guide molecule, typically labeled DNA molecules, but not necessarily It is the DNA obtained from the patient or the target sample to be analyzed. The labeling of the labeled DNA molecule can be of any type known to those of ordinary skill in the art including, but not limited to, fluorescent and radioactive labels. The labeling may also consist of one to five atoms which are significantly heavier than the average molecular weight of the DNA, as disclosed in U.S. Patent No. 7,291,468 to U.S. Patent No. 7,291,467, U.S. Patent No. 7,288,379, and WO2006/019903. Further teachings regarding the labeling are incorporated herein by reference in their entirety. In some specific cases, the guide molecule is an unlabeled DNA molecule, preferably it is not derived from a patient or target sample, but should have similar physical properties (length, width, density, and / or other features). The guide molecule may or may not have a binding force associated with the labeled molecule, such as by a sequence-specific or non-sequence-specific combination. The use of unlabeled nucleic acid molecules provides another means of controlling high density barriers. Unlabeled nucleic acid molecules and labeled nucleic acid molecules are passed through &amp; 5. As the percentage of unlabeled nucleic acid molecules in the mixture increases, the labeled nucleic acid molecules will, on average, become more and more dispersed (once attached to 26 200912307 on the surface) 'sub-reduced two labeled nucleic acid molecules will phase Neighboring and/or touching can be this.忒 control is valuable because the indicated nucleic acid molecules have a minimum distance' may be preferred for increasing the effectiveness of the interpretation system for labeling. For example, if two labeled molecules are adjacent to each other, the 轱η of one nucleic acid molecule may or may not be distinguishable from the label on the other nucleic acid molecule. In addition, the natural properties of multi-stranded nucleic acid molecules self-organizing, when the nucleic acid molecules are completely adjacent to each other, will create greater homogeneity in the molecular arrangement. With the specific facts of the labeled and unlabeled nucleic acid molecules, even after targeting the density of attachment points on the surface to be almost zero-high density intervals, and thus achieving high homogeneity, Isolation of nucleic acid molecules. Although the sample nucleic acid molecule is typically displayed prior to mixing with the unlabeled nucleic acid molecule, it can be in any particular case at any point in the process of the method, in the first round of the method of the invention. The guide molecules are attached and arranged in the matrix region. Subsequently, a second round is performed to attach and align the analytes around the guide molecules in the same matrix region; The directional molecule can be quite long, for example, hundreds of thousands of test pairs in the active double strands to maximize the effectiveness and uniformity of the molecular alignment step. The guide molecule is attached along the entire length of the guide molecule so as to prevent it from being separated by the analyte fraction = alignment force during the first / 〇 W period. Usually, the guide molecules are not labeled to avoid the period of analysis after the alignment (5), and it is confused with the analyzed molecules of the standard. However, the labels of the analyte molecules can be used differently: the standard δ has been equipped with guide molecules to allow The wizard's decision is to locate the analyte molecule. The guide molecule can act as a 87-phase or channel edge on the surface of the substrate to improve the alignment of the analyte molecules (see also Figure 13). As for the substrate having the film region, the attachment may occur in or near the film region, but the alignment and spacing control should be substantially in the film region of the substrate. In other words, the attachment zone can comprise both a film zone and another adjacent zone of the substrate. In some specific cases, the substrate has only attachment regions. In some specific instances, the matrix has both an attachment zone and an alignment zone. The matrix of only the attachment zone is illustrated in Figure 12. In this particular case, attaching one end of each analyte molecule to the substrate will allow for attachment and alignment of both regions. The size of the substrate region, the length of the analyte molecule, and the target spacing of the nucleic acid molecules determine the amount of nucleic acid molecule in the liquid that is in contact with the substrate. The longer the molecule, the wider the target interval required to minimize the molecular overlap downstream of the alignment force. Step #ι, controlled attachment of the numerator-end and/or linker as described above. Steps #2 and #3, the molecular arrangement and attachment of the intact molecules were also carried out as described above. The nucleic acid can be attached to any region on the substrate. In some specific cases, the number of linkers or contacts limits the amount of nucleic acid molecule added during the attachment reaction. Limiting the amount of unreacted linker is appropriate because unreacted linker molecules can still be used to bind the aligned molecules and interfere with the alignment reaction. In some specific instances, the attachment region includes both a linker (for terminating the nucleic acid) and a substrate modification (for binding to the aligned nucleic acid). The substrate having both the attachment and alignment regions is illustrated in Fig. 2, wherein the arrows indicate the alignment regions in the '' direction. The density of the aligned molecules can be changed as the direction of flow changes (see also below). It can be used in a technical matrix that may be fully or partially functionalized. For example, 28 200912307 Techniques and reagents known in the art to functionalize the surface of a substrate to create a hydrophobic surface (see, for example, Allemand, Bensimmin, et al., "PH-value-dependent specific binding and combing of DNA

Binding and Combing 〇f DNAr% Biophysical Journal, 1997 年10月）。疏水性表面在進行該方法之期間，藉著促進各種 鹽類、蛋白質等等之移除（例如藉著洗去），使其遠離附接在 基質上的核酸分子，而發揮一類型清洗劑之作用。疏水性 表面亦在利用一般技藝人士已知的凹液面方法之一排列 時，藉著使親水性污染物留在溶液中而擔任清洗劑。疏水 性表面仍允許腦的結合，藉著使疏水性表面官能化及/ 或藉著使在表面上的DNA脫水。在某些具體事實中，該疏 水性表面是無定形碳、石墨或類似的表面。可在附接核酸 分子之前或之後，例如在附接連接子之前或之後，將基質 表面官能化。在國際專利公開案W02007/076132中描述了 核酸附接的額外觀點。 核酸分子的分子排列Binding and Combing 〇f DNAr% Biophysical Journal, October 1997). Hydrophobic surfaces serve as a type of cleaning agent during the course of the process by facilitating the removal of various salts, proteins, and the like (eg, by washing away) away from the nucleic acid molecules attached to the substrate. effect. The hydrophobic surface also acts as a cleaning agent by leaving the hydrophilic contaminants in solution when aligned by one of the methods known to those skilled in the art. The hydrophobic surface still allows binding of the brain by functionalizing the hydrophobic surface and/or by dehydrating the DNA on the surface. In some specific instances, the hydrophobic surface is amorphous carbon, graphite or a similar surface. The surface of the substrate can be functionalized before or after attachment of the nucleic acid molecule, such as before or after attachment of the linker. Additional views of nucleic acid attachment are described in International Patent Publication No. WO2007/076132. Molecular arrangement of nucleic acid molecules

V 、_本發明之方法的第二個步驟是使用許多技術的任一種 進仃分子排列’典型地包括但不限於流體流動，並可視需 要包括鹽濃度和溫度控制°附接在基質表面之末端以外的 核酸分子部分（例如多股秒赌八2X+ 夕奴核馱分子），會隨著流體流動的方向 與流體-起移動，纟因此導致附接在基質那一點下游的伸 直。核酸分子之自我·組織特性亦會使其等在基質表面上的 部分單層十，以實質上平杆的士 4 μ 工扪 仃的方式排列，藉此協助分 分子排列。 刀丁 07 29 200912307 欲在排列後達到經靶定之高密度間隔，優先使流體移 動的運動特徵配合核酸分子特性和該方法第一個步驟（附接 至基質）的可控制因素，以創造經靶定之結果。運動特徵是 可改變的，包括但不限於：流體的速度、速率或加速度， 改變流體流動的速度、速率或加速度、流體流動的方向性、 流體之體積和流體流動的期間。V, _ The second step of the method of the invention is to use any of a number of techniques to introduce a molecular arrangement 'typically including but not limited to fluid flow, and optionally including salt concentration and temperature control ° attached to the end of the substrate surface The portion of the nucleic acid molecule other than the molecule (e.g., a multi-strand second gamma 2X+ nucleus molecule) moves with the fluid in the direction of fluid flow, thereby causing the extension to be attached downstream of the substrate. The self-tissue properties of the nucleic acid molecule also cause a portion of the monolayer 10 on the surface of the substrate to be arranged in a substantially flat rod of 4 μg to assist in molecular alignment. Knife 07 29 200912307 To achieve a targeted high-density interval after alignment, prioritize the movement characteristics of the fluid to match the characteristics of the nucleic acid molecule and the controllable factors of the first step of the method (attached to the matrix) to create the target The result. The motion characteristics are changeable, including but not limited to: the velocity, velocity or acceleration of the fluid, the speed, rate or acceleration of the fluid flow, the directionality of the fluid flow, the volume of the fluid, and the period of fluid flow.

所使用之流體可包括，但不限於：極性溶劑，如水或 酒精；非-極性溶劑，如丙蜩；或氣體，如空氣、氮氣或氬 氣。亦可在排財驟期間的任何時間並以任何比例改變流 體類型。流體可按指定量含有添加物，包括但不限於：一 或多種鹽類、一或多種濕潤劑，及/或一或多種影響表面張 力及/或結合特性的材料。可在排列步驟期間的任何時間並 以任何比例改變流體添加物。 特徵’亦可在排列步驟期間 變。亦可使用流體運動特徵、 體溫度的組合來進行該步驟。 流體溫度是另一個可控制的 的任何時間並以任何比例改 流體類型、流體添加物和流The fluid used may include, but is not limited to, a polar solvent such as water or alcohol; a non-polar solvent such as propylene; or a gas such as air, nitrogen or argon. The fluid type can also be changed at any time during the treasury and at any rate. The fluid may contain additives in specified amounts including, but not limited to, one or more salts, one or more wetting agents, and/or one or more materials that affect surface tension and/or bonding characteristics. The fluid additive can be changed at any time during the alignment step and at any ratio. The feature ' can also vary during the arranging step. This step can also be performed using a combination of fluid motion characteristics and body temperature. The fluid temperature is another controllable at any time and changes in any proportion of fluid type, fluid additive and flow

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排列之分子（包括嚮導分子）结人。择您A 刀丁厂。口祆戴會多少影響分子在定 向流體流動(或其他所施加之排列力，如電或磁場)中的行 為，並促使分子排列。標籤可結合在分子未與基質附接的 末端’及/或在沿著分子長度的一或多 夂夕個點上。選擇標籤的 尺寸、形狀和材料組合，以便與該 之其他7L件及直所 有變化一起工作。使標籤結合的方 ’、 扪万去為—般技藝人士已知 的0 30 200912307 該方法步驟的另-變化，是使用電場作為定向力以廣 泛地排列分子，通常是在流動通道中（參見例如pcT經公開 之申請案W〇2〇〇7/〇76132 A2)。可在流體流動以外或之外， 有或無標籤之下使用電場。流動通道由忌水壁和親水膜的 下表面構成。流動通道可以有或可以沒有上表面。—旦分 子的剩餘部分普遍地已經伸直，經附接之末端便會停止分 子運動，並以電場的方向排列。然後以遞減鹽濃度之水溶 液沖洗普遍經排列之分子。降低之鹽漢度降低了鹽.引起之 七“夂基團排斥力的鈍化。這硬化並弄直個別的多股分子， 因為在個別分子内碟酸基團移動分開得更遠。增加磷酸基 團之排斥，亦增加了在個別分子之間的排斥力。可利用鹽 濃度（物種和濃度兩者）調節該排斥影響，以調節隨之而來在 經排列分子之間的間隔。可使用該電場方法的其他變化。 實例僅企圖解釋，並無意限制對本發明預見的技術。 邊方法步驟的另—變化是在流體流動以外或之外，使 用磁f生作為疋向力，以進行分子排列。可在流體流動以外 或之外，有或沒有標籤之下使用之。使用磁性之技術的實 例是以—般技藝人士已知的方法，包括但不限於使用磁性 和附接金屬奈米-顆粒之生物學分離或純化技術的方法，將 f屬或磁性標鐵或小珠附接在分子上的-或多個位置。在 八中以止圖排列之方向投注磁力，會在流體中拉動 在：子上的金屬標鐵朝向磁鐵，但因為末端附接在基質上 而停止° &amp;會產生或增強弄直和排列的影響。可使用該磁 去的其他變化。實例僅企圖解釋，並無意限制對本發 31 200912307 明預見的技術。 本發明亦包括另外的排列技術。在一具體事實中，藉 著分子梳進行排列’其中並沒有流動通道，但將基質浸在 溶液中，允許雜交或另行反應，並以經控制之方向拉離溶 液（參見美國專利第6,458,255號）。 在本方法之第一個步驟中使用的材料和技術，連同其 所有的變化和組合，均可用來在排列期間拉伸多股核酸分 子’並引起其等部分或全部從核酸分子的螺旋或其他天然 彎曲構型中解開，並形成核酸分子的，，梯狀，，構形（Li， 1999.在生物學中的掃描探針顯微分析（STM/AFM)和應用 (Scanning probe microscopy(STM/AFM) and applications in biology) Applied Physics A. 68, 225-258 ;亦參見圖 13)。在 本方法的第三個步驟中描述之將經排列核酸分子固定在基 質上的技術，可能有助於梯狀構形的創造或維持。核酸分 子的梯狀構形對於使用核酸分子之直接顯影，尤其是藉= 在dsDNA上原子標記之TEM直接顯影的任何分析會是^另^ 有益的。核酸分子的梯狀構形有助於影像詮釋，及/或改良 影像詮釋的品質，因為在與基質表面同—水平中的標記^ 接近，但*會彼此掩蓋。在某些具體事實中，藉著:經排 ，之分子與特殊表面結合而產生”梯狀，，構形。在某些具體事 貫中’該表面為疏水性表面。在某4b 1 卞一吳體事實中，該表面 為石墨表面。纟第二個步驟中使用的材料和技術，連㈣ 所有變化和組合，亦可在排列期間用以預防或另 ： 股核酸分子的明顯拉伸。 夕 32 200912307 使經排列之分子與基質表面結合 可以被動模式或經由主動模式，將經排列之分子與基 質結合。以被動模式，在形成排列後，經排列之分子可結 合於基質表面。在該方法中’與表面之結合是非_共價的。 在某些具體事實中，該結合為可逆的。這可藉著下列之一 或組合達成：使定向之流體流動減緩及/或停止、改變流體 及/或環境的溫度、在流體流動停止之後以定向之方式使分 子脫水’以及在移除流體之前或之後，加入促進與表面之 結合的試劑及/或活化劑。由各種因素控制經排列分子與表 面的結合，包括溶劑組成和表面特徵。在某些具體事實中， 基質的排列區會具有與附接區不同的特徵，結果使經排列 分子選擇性地結合在基質之排列區上。在某些具體事實 中，基質沒有分開的附接區，而經排列之分子會與附接該 分子之末端的相同區域結合（例如，參見圖12)。在這類具 體事實中，可修飾基質的表面，以允許附接分子的末端（例 如經由連接子）並與經排列之分子結合兩者。 在主動模式中，進行反應以附接經排列之分子至表 面在某些具體事實中，修飾該表面以便與經排列之分子 反應。在某些具體事實中，修飾該表面以便選擇性地與經 排列分子之主鏈反應，但不與經排列分子的末端反應。將 核酸分子與表面附接的反應，為在技術領域中已知的（參見 例如W02007/076i32)。本發明包括任何將經排列之分子與 表面附接的方法。 在某些具體事實中，當使DNA與表面結合時，延伸該 33 200912307 DNA。延伸核酸分子的方法為在技術領域中已知的，例如 降低周圍離子強度會導致增加分子内的排斥，以及核酸的 延伸。 在某些具體事實中，結合亦包括將經排列之分子固定 在，質表面上。在某些具體事實中，所有的分子均附接在 土貝表面上纟排列之後將分子附接到基質表面時，可使 用熟諳此藝者已知的方法’包括：uv_交聯、在麟酸基團與 官能化表面(例如胺基基團等等)之間的離子吸引力、使用經 i $之DNA以結合其他的表面修飾（例如塗在基質上的 氮）、陽離子表面處理，或依賴凡得瓦爾力。在國際專利公 開案W〇2007/076l32中描述了核酸附接的額外觀點。 重複方法步驟 一在本發明方法的某些應用中，可對相同的基質重複第 -、第二和第三個步驟多次。可改變所進行之步驟的順序 和次數。在一變化Φ，-P 4 , Μ 中可進订苐一個步驟（附接核酸分子的 一端），以經控制之圓安收 lL ^ 户 圖案將一些DNA分子附接到基質上。然 後進行第二和第二伽、 —個步驟（排列並結合經排列之核酸分 子)，以排列這些分子，並可視需要將其等固定在基質上。 然後再次進行第—個步驟，將額外的DNA分子附接到相同 的基質上’或在相同附接區中之分子的第一個部分單層 ’、’、後再度進仃第二和第三個步驟。該重複步驟方法的 較佳具體事實，_ I&amp; «者進仃第一、第二和第三個步驟，在基 質上以高密度間隔之圖案，附接、排列並固定未經標示之 撕A分子(例如嚮導分子)。使用固定方法，使得未經標示 34 200912307 刀子在/¾:於流體中時不會脫離。隨後在相同的附接 區中進仃經標不DNA分子的末端-附接和排列（第—和第二 個步驟）。在第二個步驟期間’在表面上經排列之未經標示 勺DNA刀子（例如嚮導分子”擔任改良經標示之dna分子 排列的指弓卜在一端的附接可發生在基質的開放空間中， 或在未經標示DNA分子之部分單層的上方。然後對經標示 2财分子進行第三個步驟。在某些變化中，未經標示之 向$刀子的咼岔度間隔大得足以容許經標示之dna分子在 ，導分子之間排列’並與表面結合，肖嚮導分子—起形成 早層。在其他變化中’會在未經標示之嚮導分子的上方附 接及/或排列一些或全部的經標示Dna分子，形成二或多 層有些變化可使用形成許多層經排列分子的方法。在這 —I化的任一種中’附接區可以是與每個第一個步驟相同 或不同的，且實際的附接及/或排列可發生在基質表面上或 在其他分子的上方。 藉著以下的實施例進一步解釋本發明，不應將其解釋 為更多的限制。特別將所有在本中請案中引用之參考文獻 (/括文獻、經發證之專利、經公開之專利中請案和同在申 凊中之專利中請案）的完整内容，以弓丨用方式納人本文中， 特別是在上文令提及的教示。 實施例 實施例1 本實施例描述使用上述的方法，在得自欲分析之試樣 的dsDNA(試樣dsDNA)之高密度間隔的部分單層中進行分 35 200912307 子排列。以具有可檢測之分子量（與在得自穿透式電子顯微 鏡（TEM)之影像上的背景雜訊對比）的原子來標示試樣 dsDNA分子。該部分單層亦含有得自與目標試樣不同來源 的未經標示之dsDNA，其隔開經標示之dsDNA，以改良分 子排列之品質，以及試樣dsDNA分子的後續判讀。使用該 較佳具體事實的分析’以便在相鄰的判讀（較佳的是具有 5,000或更多，或10，_或更多，《兩萬(2〇,議)或更多個 驗基對）中，判定DNA序列。亦利用該方法，藉著使用在本 方法三個步驟中的技術變化，定序更長和更短的DNA雙股。 步驟一： 在第個步驟中，以經控制之圖案，將多個dsDNA分 子的端附接在基質上。該基質具有一或多個薄膜區（厚度 小於3 0奈米），其中舍南丨田 、會引用TEM使目標試樣dsDNA顯影。 ❼十薄膜材料，以便在影像中引起最少的背景雜訊。選擇 I接！圖20案使所產生之—ΝΑ分子在排列後具有低於大The aligned molecules (including the guide molecules) are connected. Choose your A knife factory. Mouth wear affects the behavior of molecules in a directed fluid flow (or other applied alignment force, such as electricity or magnetic fields) and promotes molecular alignment. The tag can bind to the end of the molecule not attached to the substrate&apos; and/or at one or more points along the length of the molecule. Select the size, shape, and material combination of the label to work with the other 7L pieces and all other variations. The combination of the label's ', 扪 去 为 — 0 0 0 0 0 0 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 pcT is published in the application W〇2〇〇7/〇76132 A2). The electric field can be used with or without a label outside or outside the fluid flow. The flow channel is composed of a water-repellent wall and a lower surface of the hydrophilic membrane. The flow channel may or may not have an upper surface. Once the remainder of the molecule has been generally straightened, the attached end stops the molecular motion and is aligned in the direction of the electric field. The generally aligned molecules are then rinsed with an aqueous solution of decreasing salt concentration. The reduced salt degree reduces the salt. The resulting "passivation of the ruthenium group's repulsive force. This hardens and straightens out individual molecules, because the acid groups in the individual molecules move apart further. Increase the phosphate group The rejection of the group also increases the repulsive force between individual molecules. The salt concentration (both species and concentration) can be used to adjust the repulsive effect to adjust the spacing between the aligned molecules. Other variations of the electric field method. The examples are merely intended to be illustrative and are not intended to limit the techniques foreseen by the present invention. Another variation of the method steps is to use magnetic f-force as a directional force in addition to or outside the fluid flow for molecular alignment. It may be used in addition to or in addition to fluid flow, with or without a label. Examples of techniques using magnetic are methods known to those skilled in the art including, but not limited to, the use of magnetic and attached metal nano-particles. A method of biological separation or purification technique in which a f-genus or a magnetic iron or a bead is attached to - or a plurality of positions on a molecule. The metal is pulled in the body toward the magnet, but because the end is attached to the substrate, the stop &amp; will produce or enhance the effect of straightening and alignment. Other variations of the magnetic removal can be used. It is not intended to limit the technology foreseen by the present invention. The present invention also includes an additional arrangement technique. In a specific fact, the arrangement is by a molecular comb, in which there is no flow channel, but the substrate is immersed in the solution, Allow hybridization or additional reaction and pull the solution in a controlled direction (see U.S. Patent No. 6,458,255). The materials and techniques used in the first step of the process, along with all variations and combinations thereof, can be used Stretching multiple strands of nucleic acid molecules during alignment and causing them to partially or completely unravel from the helical or other naturally curved configuration of the nucleic acid molecule, and form a ladder, conformation of the nucleic acid molecule (Li, 1999. Scanning probe microscopy (STM/AFM) and applications in biology Applied Physics A. 68, 225-258; see also Figure 13). The technique of immobilizing aligned nucleic acid molecules on a substrate as described in the third step of the method may contribute to the creation or maintenance of ladder conformations. Any configuration that is directly developed using nucleic acid molecules, especially by TEM directly on the dsDNA, may be beneficial. The ladder configuration of the nucleic acid molecule facilitates image interpretation, and / Or improve the quality of image interpretation, because the mark ^ in the same level as the surface of the substrate is close, but * will cover each other. In some specific facts, by: the platoon, the molecules combined with the special surface to produce the ladder Shape, configuration. In some specific cases, the surface is a hydrophobic surface. In the case of a 4b 1 卞 吴 body, the surface is a graphite surface. The materials and techniques used in the second step, in addition to (iv) all changes and combinations, can also be used during the alignment to prevent or otherwise significantly stretch the stranded nucleic acid molecules.夕 32 200912307 Combining Aligned Molecules with the Surface of the Substrate The aligned molecules can be bound to the matrix either in a passive mode or via an active mode. In the passive mode, after the alignment is formed, the aligned molecules can be bonded to the surface of the substrate. In this method, the combination with the surface is non-covalent. In some specific cases, the combination is reversible. This can be achieved by one or a combination of slowing and/or stopping the directed fluid flow, changing the temperature of the fluid and/or environment, dehydrating the molecules in an oriented manner after the fluid flow ceases, and before removing the fluid Or afterwards, an agent and/or an activator that promotes binding to the surface is added. The combination of aligned molecules and surfaces, including solvent composition and surface characteristics, is controlled by a variety of factors. In some specific cases, the aligned regions of the matrix will have different characteristics than the attachment regions, with the result that the aligned molecules are selectively bound to the aligned regions of the matrix. In some specific instances, the matrix has no separate attachment regions, and the aligned molecules will bind to the same region to which the ends of the molecules are attached (see, for example, Figure 12). In such specific facts, the surface of the substrate can be modified to allow attachment of the ends of the molecule (e.g., via a linker) and binding to both of the aligned molecules. In the active mode, a reaction is performed to attach the aligned molecules to the surface. In some specific instances, the surface is modified to react with the aligned molecules. In some specific instances, the surface is modified to selectively react with the backbone of the aligned molecules, but not with the ends of the aligned molecules. The reaction of attaching a nucleic acid molecule to a surface is known in the art (see for example W02007/076i32). The invention includes any method of attaching aligned molecules to a surface. In some specific cases, when the DNA is bound to the surface, the 33 200912307 DNA is extended. Methods of extending nucleic acid molecules are known in the art, for example, reducing peripheral ionic strength results in increased intramolecular repulsion, as well as elongation of nucleic acids. In some specific cases, the combination also includes immobilizing the aligned molecules on the surface of the material. In some specific cases, when all molecules are attached to the surface of the soil, the molecules are attached to the surface of the substrate, and the method known to the artist can be used to include 'uv_crosslinking, in the Ionic attraction between acid groups and functionalized surfaces (eg, amine groups, etc.), use of DNA of i $ to bind other surface modifications (eg, nitrogen coated on a substrate), cationic surface treatment, or Rely on Van Der Valli. Additional views of nucleic acid attachment are described in International Patent Publication No. 2007/076l32. Repeat Method Step 1 In some applications of the method of the invention, the first, second and third steps can be repeated multiple times for the same substrate. The order and number of steps performed can be changed. A step (attaching one end of the nucleic acid molecule) can be made in a change Φ, -P 4 , Μ, and some DNA molecules are attached to the substrate by a controlled round-up lL ^ user pattern. The second and second gamma steps are then performed (arranged and combined with the aligned nucleic acid molecules) to align the molecules and immobilize them on the substrate as needed. Then the first step is performed again, attaching additional DNA molecules to the same substrate 'or the first part of the molecule in the same attachment zone', and then re-entering the second and third Steps. A preferred specific fact of the repetitive step method, _I&amp; «Into the first, second and third steps, attaching, arranging and fixing the unlabeled tear A on the substrate in a pattern of high density intervals Molecules (such as guide molecules). Use a fixed method so that the unmarked 34 200912307 knife will not detach when it is in the fluid. The ends-attachment and alignment of the labeled DNA molecules are then carried out in the same attachment region (first and second steps). During the second step, the attachment of an unmarked DNA knife (eg, a guide molecule) arranged on the surface to the modified labeled molecular molecule at one end may occur in the open space of the substrate, Or above a single layer of unlabeled DNA molecules. Then perform the third step on the labeled 2 molecules. In some variations, the unmarked spacing to the $knife is large enough to allow The labeled dna molecules are arranged between the guide molecules and bind to the surface, and the Schottky molecules form an early layer. In other variations, 'they will be attached and/or arranged some or all of the unlabeled guide molecules. The labeled Dna molecule, which forms two or more layers, can be used to form a plurality of layers of aligned molecules. In either of these, the 'attachment zone can be the same or different from each of the first steps, And the actual attachment and/or alignment may occur on the surface of the substrate or above other molecules. The invention is further explained by the following examples and should not be construed as limiting. All the references cited in this application (including the documents, the patents issued, the patents in the published patents, and the patents in the application) are all in the form of bows. The teachings mentioned herein, in particular, are mentioned above. EXAMPLES Example 1 This example describes the use of the above method for high density separation of dsDNA (sample dsDNA) from samples to be analyzed. The partial monolayer is divided into 35 200912307 sub-arrangements. The sample dsDNA molecules are labeled with atoms with a detectable molecular weight (compared to background noise from an image obtained from a transmission electron microscope (TEM)). The monolayer also contains unlabeled dsDNA from a different source than the target sample, which separates the labeled dsDNA to improve the quality of the molecular arrangement and subsequent interpretation of the sample dsDNA molecule. Analyze 'to determine the DNA sequence in an adjacent interpretation (preferably having 5,000 or more, or 10, _ or more, 20,000 (2), or more than one test pair). Also use this method, by using it in this The technical changes in the three steps of the process, sequencing the longer and shorter DNA double strands. Step 1: In the first step, the ends of the plurality of dsDNA molecules are attached to the substrate in a controlled pattern. The substrate has one or more thin film regions (thickness less than 30 nm), wherein Shenan 丨田, TEM is used to develop the target sample dsDNA. ❼10 film material to cause minimal background noise in the image. I pick up! Figure 20 makes the resulting ΝΑ molecules have lower than large after alignment

，，勺一十（）奈米的高密度間隔測量值。% + v &amp; 里值藉者在液體中以所選 擇之街度 合萬能的連接子 子），獲得該經控制之分接任何驗分子的連接 盥，其“ 刀子間隔。然後使這些萬能的連接子 入在基質上的一或多個經限 基質之薄臈的特定部分。 附接區接觸。附接區覆蓋 子的觸之前’可視需要製備附接區，以促進連接 連接子的結合。在基質上對所選擇卜以：低或防止 十版印刷技術與感光性試劑之組合。在液體 36 200912307 中之連接子與基質接觸的期間内，擴散會使其等大約均句 地分散，隨後在那裡藉著共價力而允許或引起其等與表面 結合。然後移除任何未與表面結合的連接子。 、 在將連接子固定於基質上之後，使dsDNA分子與連接 子、’α σ以與所使用之萬能連接子互補的方法製備排列步 驟。該結合之方法為一般技藝人士已知的，並包括但不限 2直接共價鍵結，或在連接子上的萬能單股核苷酸序列標 籤’其⑬者與纟dsDNA分子上互補❸單股標藏雜交而結 合。dsDNA分子的長度可能是多變的，而僅受限於在其上 發生排列之基質表面積的尺寸。在本具體事實中，心崎 分子是經標示之目標試樣dsDNA和得自其他來源之未經標 示dSDNA的混合物。按照歡之計畫，目標試樣dsDNA^ 個別的驗基上有標示，其在藉由高解析tem拍攝之影像中 是可以區別的，如在美國專利第7,291，468號中描述的標 不。未經標示之dsDNA對經標示之dsDNA以至少二比— ㈤)之比例出現在混合物中。在排列之後，未經標示之 dsDNA不再有形地遮蔽在目標試樣七顺上之標記的影 像’並達到使經標示之_财分子彼此分離的目的（作為間 隔基）’改善了標記的證釋。預期影像的發釋和分析會允許 判定高比例之目標試樣dsDNA的序列。 ° 步驟二： 在第二個步驟中，進行分子排列。使附接於表面之末 端以外的分子部分隨著排列力的方向移動，並因此使附接 點的下游伸直。鹽漢度、溫度和表面處理的控制，引起多 37 200912307 股核酸分子之自我·組織的特性，在表面上的部分單層中形 成平行線具有經控制之間隔。一或多個微流體通道引導 流體覆盖基皙。W4姐· Γΰ θ. y- - ^附接區疋在一或多個薄膜的上游末端，將 其設計成具有在排列後比目標試樣分子更長的長度。流體 流動可視需要與其他分子排列技術變化結合，包括但不限 於&amp;制在梯度中的鹽濃度、控制溫度，並使用電場或磁場， 在使dsDNA以流體流動之方向移動時提供額外的力。 步驟三： ^在第三個步驟中，將所有經排列之核酸分子固定在基 質表面上。這可藉著使定向之流體流動減緩、當流體流動 停止時以定向方切dsDNA+子脫水，並在移除流體之後 加入與表面產生永久結合之試劑及/或活化劑的組合而達 成。其他將分子固定在基質表面上的方法為熟諳此藝者已 知的。 圖5解釋了與混合未經標示和經標示之分子，以便在 排列之後選擇性地分離經標示分子組合之高密度間隔的結 果。圖5與圖3相同，但改變了 DNA分子的明暗，以表示 混合經標示和未經標示之分子的影響。 實施例2 根據例行的技術，產製具有序列A和序列B之寡核苷 酸連接子的基質。根據下文的實施例3製備未經標示之5〇处 嚮導分子，結果產生具有與序列A互補之黏性末端的嚮導 分子。隨後將這些嚮導分子加至基質中，結果產生嚮導分 子與具有序列A之連接子的附接。藉著使流動流體流過基 38 200912307 質上，使嚮導分子相鄰排列。在下一個步驟中，藉著移除 流動流體使經排列之嚮導分子與基質結合，產生如在圖i 3 中敘述的單層。 根據下文的實施例3製備經標示之8-1 6kb分析物分 子，結果產生具有與序列B互補之黏性末端的分析物分子。 隨後將這些分析物分子加至基質中，結果產生分析物分子 與具有序列B之連接子的附接。該分析物分子的後續排列 以及與基質之結合’產生其中該分析物分子落在由嚮導分 (' 子建立之格子内的圖案（參見圖13)。 實施例3,, spoon a ten () nanometer high-density interval measurement. % + v &amp; l value borrower in the liquid with the selected street degree omnipotent linker), obtain the controlled connection of any check molecule, its "knife interval. Then make these universal connections A specific portion of the thin layer of one or more bounding substrates that are sub-into the substrate. Contact area contact. The attachment area covers the contact area before the contact area can be prepared to facilitate the attachment of the connecting link. The substrate is selected to: low or prevent the combination of the ten-printing technique and the photosensitive agent. During the contact of the linker in the liquid 36 200912307 with the substrate, the diffusion causes it to be dispersed approximately uniformly, followed by Where it allows or causes it to bind to the surface by covalent forces. Then remove any linker that is not bound to the surface. After fixing the linker to the substrate, the dsDNA molecule and the linker, 'α σ The alignment step is prepared in a manner complementary to the universal linker used. The method of combining is known to those of ordinary skill in the art and includes, but is not limited to, direct covalent bonding, or a versatile single on the linker. The nucleotide sequence tag '13' binds to the complementary single-stranded hybridization of the 纟dsDNA molecule. The length of the dsDNA molecule may be variable, but only limited by the size of the surface area on which the matrix is aligned. In this specific fact, the Minzaki molecule is a mixture of the labeled target sample dsDNA and unlabeled dSDNA from other sources. According to Huan's plan, the target sample dsDNA^ is marked on the individual test basis. It is distinguishable in images taken by high resolution tem, as described in U.S. Patent No. 7,291,468. Unlabeled dsDNA is at least two to five ratios to the labeled dsDNA. Appears in the mixture. After the alignment, the unlabeled dsDNA no longer tangibly obscures the image of the mark on the target sample VII and achieves the purpose of separating the labeled molecules from each other (as a spacer). The proof of the mark is improved. It is expected that the release and analysis of the image will allow the determination of the sequence of the high proportion of the target sample dsDNA. ° Step 2: In the second step, perform molecular alignment. The molecular moiety other than the end moves in the direction of the alignment force, and thus the downstream of the attachment point is straightened. The control of salt, temperature and surface treatment causes the self-organizing properties of the multi-drug molecules of 2009. Parallel lines formed in a portion of the monolayer on the surface have controlled spacing. One or more microfluidic channels direct the fluid to cover the substrate. W4 sister · Γΰ θ. y - - ^ Attachment zone 疋 in one or more thin films The upstream end is designed to have a longer length than the target sample molecule after alignment. Fluid flow may be combined with other molecular alignment techniques, including but not limited to &amp; salt concentration in the gradient, temperature control, And using an electric or magnetic field, it provides additional force when moving the dsDNA in the direction of fluid flow. Step 3: ^ In the third step, all aligned nucleic acid molecules are immobilized on the surface of the substrate. This can be achieved by slowing the flow of the directed fluid, dewatering the oriented DNA strands as the fluid flow ceases, and adding a combination of reagents and/or activators that permanently bind to the surface after removal of the fluid. Other methods of immobilizing molecules on the surface of a substrate are known to those skilled in the art. Figure 5 illustrates the results of mixing high density partitions with unlabeled and labeled molecules to selectively separate labeled combinations of molecules after alignment. Figure 5 is the same as Figure 3 but with the change in the light and dark of the DNA molecules to indicate the effect of mixing the labeled and unlabeled molecules. Example 2 A substrate having an oligonucleotide linker of sequence A and sequence B was produced according to the conventional technique. An unlabeled 5 向导 guide molecule was prepared according to Example 3 below, resulting in a guide molecule having a viscous end complementary to Sequence A. These guide molecules are then added to the matrix, resulting in the attachment of the guide molecule to the linker with sequence A. The guide molecules are arranged adjacent to each other by flowing the flowing fluid through the base. In the next step, the aligned guide molecules are combined with the matrix by removing the flowing fluid to produce a single layer as described in Figure i3. The labeled 8-16 kb analyte molecules were prepared according to Example 3 below, resulting in an analyte molecule having a viscous end complementary to sequence B. These analyte molecules are then added to the matrix, resulting in the attachment of the analyte molecule to the linker with sequence B. Subsequent alignment of the analyte molecules and binding to the substrate produces a pattern in which the analyte molecules fall within the grid created by the guides (see Figure 13).

為了標示而製備DNA 使用 HydroShear® 機器（Digilab Genomic Solutions, Ann Arbor，MI)打碎基因組DNA，產生長度大約8,000到 1 6,000個鹼基對的DNA。使用End-ItTMDNA末端-修理套組 (Epicenter Biotechnologies, Madison, WI)，進一步加工 DNA ’以移除單股突出物。DNA的加工，結果產生具有鈍 端之DNA，其為5’磷酸化的。在下一個步驟中，以適體 (adaptamer)裝配DNA。使用下列的引子製備適體： 引子ZS1 5’ GGA CGG TCT TCC CAG TCA CGA CAC TGT GTA GAA CGA CGG CCT GAG 3，（SEQ ID ΝΟ:1)Preparation of DNA for labeling Genomic DNA was disrupted using a HydroShear® machine (Digilab Genomic Solutions, Ann Arbor, MI) to produce DNA of approximately 8,000 to 1 6,000 base pairs in length. DNA was further processed using the End-ItTM DNA End-Repair Kit (Epicenter Biotechnologies, Madison, WI) to remove single-strand protrusions. Processing of the DNA results in a blunt-ended DNA that is 5&apos; phosphorylated. In the next step, the DNA is assembled with an adaptor. The aptamer was prepared using the following primers: Primer ZS1 5' GGA CGG TCT TCC CAG TCA CGA CAC TGT GTA GAA CGA CGG CCT GAG 3, (SEQ ID ΝΟ: 1)

弓I子ZS1RCBow I ZS1RC

3’ CC AGA AGG GTC AGT GCT GTG AGA CAT CTT3' CC AGA AGG GTC AGT GCT GTG AGA CAT CTT

GCT GCC GGA CTC 5，（5’ CTC AGG CCG TCG TTC TAC 39 200912307 AGA GTG TCG TGA CTG GGA AGA CC 3’ SEQ ID NO:2)。引子ZS IRC在5’端上具有磷酸鹽。 以等量將ZS1和ZS1RC引子混合在一起，加熱至95 °C，並慢慢地冷卻至4°C，結果產生在一邊具有未經磷酸化 之5’GGAC突出物的適體，而該適體在另一邊以5’磷酸鹽 使末端鈍化。隨後將該適體連接到DNA片段中，使用過量 很多的適體，結果使適體之鈍端與具有鈍端之基因組DNA 片段連接。連接的最後結果為DNA片段的混合物，具有在 8kb到1 6kb之間的長度，並在每一端都具有額外45個鹼基 對之適體。過量的適體防止多個基因組DNA片段彼此連接。 DNA片段之標示 使用擴大反應標示DNA片段。使用ZS1RC作為引子， 並使用碘-U代替dTTP，結果在前進股上***了經標示之U 來代替T。使用下列組份來製備擴大混合物： 體積（微升） 試劑 濃度 2.5 10X緩衝溶液，不含Mg2+ 0.85 MgCl 50mM 0.9 dNTPs 每種 dNTP 10mM 0.5 前進引子（ZS1RC) 10mM 19.25 欲標示之DNA 1 聚合酶 25 總計 40 200912307 以下列計劃表在熱循環器上進行擴大反應： 循環步驟 溫度°c 時間 循環次數 最初的變性 92〇C 2分鐘 1 變性 92〇C 30秒 10 黏接 62〇C 30秒 延伸 68〇C 18分鐘 最後的延伸 68〇C 7分鐘 1 4°C 〇〇 在完成擴大反應後，使用YM100 MICROCON®離心濾 器（Millipore，Billerica, MA)過濾所得的產物。 在後續的步驟中，使用ZS1 S引子標示逆向股。GCT GCC GGA CTC 5, (5' CTC AGG CCG TCG TTC TAC 39 200912307 AGA GTG TCG TGA CTG GGA AGA CC 3' SEQ ID NO: 2). The primer ZS IRC has a phosphate on the 5' end. The ZS1 and ZS1RC primers were mixed together in equal amounts, heated to 95 ° C, and slowly cooled to 4 ° C, resulting in an aptamer with unphosphorylated 5' GGAC protrusions on one side. The body is passivated on the other side with 5' phosphate. This aptamer is then ligated into the DNA fragment, using a large excess of the aptamer, with the result that the blunt end of the aptamer is ligated to the genomic DNA fragment with a blunt end. The final result of the ligation is a mixture of DNA fragments with a length between 8 kb and 16 kb and an additional 45 base pair aptamers at each end. Excess aptamers prevent multiple genomic DNA fragments from being ligated to each other. Labeling of DNA fragments The amplified reaction is used to label DNA fragments. Using ZS1RC as a primer and using iodine-U instead of dTTP, the indicated U was inserted on the advancing strand instead of T. The following components were used to prepare the expanded mixture: Volume (microliter) Reagent concentration 2.5 10X buffer solution, no Mg2+ 0.85 MgCl 50 mM 0.9 dNTPs per dNTP 10 mM 0.5 Advance primer (ZS1RC) 10 mM 19.25 DNA to be labeled 1 Polymerase 25 Total 40 200912307 Enlarged reaction on the thermal cycler with the following schedule: Cycle step temperature °c Time cycle number Initial denaturation 92〇C 2 minutes 1 Denaturation 92〇C 30 seconds 10 Bonding 62〇C 30 seconds extension 68〇C The last extension of 18 minutes 68 〇 C 7 minutes 1 4 ° C 〇〇 After the completion of the expansion reaction, the obtained product was filtered using a YM100 MICROCON® centrifugal filter (Millipore, Billerica, MA). In the subsequent steps, the ZS1 S primer is used to indicate the reverse strand.

引子ZS1S 5’ GTA GAA CGA CGG CCT GAG 3，（SEQ ID NO:3)， 引子Z1S相當於引子ZS1的18個3’端核酸。 使用得自第一個標示步驟之產物作為模板，進行第二 個標示步驟。使用ZS 1 S作為引子，結果擴大了逆向股。分 別使用溴-C和碘-U代替dCTP和dTTP。該擴大反應所得的 DNA分子，有一個鈍端和一個黏性末端，相當於ZS 1RC的 前23個核苷酸。只進行反應一次，從第一個反應的過量單 股DNA中只產生雙股DNA。使用下列組份來製備擴大混合 物： 41 200912307 體積(微升） 試劑 濃度 2.5 10X缓衝溶液，不含Mg2+ 0.85 MgCl 50mM 0.9 dNTPs 每種 dNTP 10mM 0.5 逆向引子 10mM 19.25 標示步驟1之產物 1 聚合酶 25 總計 以下列計劃表在熱循環器上進行擴大反應： 循環步驟 溫度°C 時間 循環次數 最初的變性 92〇C 2分鐘 1 變性 92 °C 30秒 1 黏接 62 °C 30秒 延伸 68〇C 18分鐘 最後的延伸 68〇C 7分鐘 0 4°C 〇〇 在完成擴大反應後，使用YM100 MICROCON®離心濾 器（Millipore，Billerica, MA)過濾所得的產物。 終產物為雙股長度的經標示DNA，在前進股上具有碘 標記，並在逆向股上有溴和碘標記，而在DNA分子的一端 具有黏性末端。 42 200912307 亦使用未經標示之核苷酸進行反應，結果產生未經標 示的DNA產物，其具有與經標示之DNA相同的長度和黏 性末端。 k斌矽氧模型生產聚-二曱基矽氧烧（pDMS)流動通道 材料 · 4”晶圓鑷子、聚苯乙烯培替氏培養皿、2支吸移管、塑 膠匙、塑膠杯、剃刀刀片、真空乾燥器、piasm〇d 〇2電漿灰 化機、鋁箔、熱板、刻度尺、提前製備的su_8模型晶圓、 PDMS和矽烧化劑（十三氟_ι，ι，2,2-四氫辛基三氯碎烷）。 程序： 將SU-8模型晶圓連同2-3滴矽烷化劑放在培替氏培養 皿中，並放在真空乾燥器中，留在真空下至少丨小時。以 按10:1(驗：固化劑）比例之組份（PDMS : SYLGARD®184得 自Dow Corning. SYLGARD是含有乙烯基基團（A部分）和氫 化矽氧烷基團（B部分）的兩部分樹脂系統）製備pDMS預-聚 物。隨後將PDMS混合物加至放在聚苯乙烯培替氏培養皿 ί 中的SU，8母晶圓中。PDMS在24小時中固化，不需加熱。 (或者可使PDMS在65°C下固化大約1-2小時；視PDMS層 的厚度而定）。從SU-8母體中剝下PDMS模型，並切成適 當的大小。 可在 David C. Duffy，J. Cooper McDonald, Olivier J.A. Schueller，和 George M_ Whitesides, “微觀流體系統在聚（二 甲基石夕氧烧）中的快速定型（Rapid Prototyping ofThe primer ZS1S 5' GTA GAA CGA CGG CCT GAG 3, (SEQ ID NO: 3), the primer Z1S corresponds to the 18 3'-terminal nucleic acids of the primer ZS1. A second labeling step is performed using the product from the first labeling step as a template. Using ZS 1 S as a primer, the result is an expansion of the reverse strand. Bromo-C and iodine-U were used instead of dCTP and dTTP. The DNA molecule obtained by this expansion reaction has a blunt end and a sticky end, which is equivalent to the first 23 nucleotides of ZS 1RC. Only one reaction is carried out, and only double-stranded DNA is produced from the excess single-stranded DNA of the first reaction. The following components were used to prepare the expanded mixture: 41 200912307 Volume (microliters) Reagent concentration 2.5 10X buffer solution, no Mg2+ 0.85 MgCl 50 mM 0.9 dNTPs per dNTP 10 mM 0.5 Reverse primer 10 mM 19.25 Product 1 of step 1 Polymerase 25 A total of the following schedules were used to expand the reaction on the thermal cycler: Cycle step temperature °C Time cycle number Initial denaturation 92〇C 2 minutes 1 Denaturation 92 °C 30 seconds 1 Bonding 62 °C 30 seconds extension 68〇C 18 Minute last extension 68 〇 C 7 min 0 4 ° C 〇〇 After completing the expansion reaction, the resulting product was filtered using a YM100 MICROCON® centrifugal filter (Millipore, Billerica, MA). The final product is a double-stranded labeled DNA with an iodine label on the advancing strand and a bromine and iodine label on the reverse strand and a sticky end at one end of the DNA molecule. 42 200912307 The reaction was also carried out using unlabeled nucleotides, resulting in an unlabeled DNA product having the same length and sticky ends as the indicated DNA. The k-bin oxygen model produces poly-dimercapto-oxygen (pDMS) flow channel materials. 4" wafer tweezers, polystyrene petri dishes, 2 pipettes, plastic spoons, plastic cups, razor blades, Vacuum dryer, piasm〇d 〇2 plasma ashing machine, aluminum foil, hot plate, scale, pre-prepared su_8 model wafer, PDMS and sputum burning agent (trifluoro-I, I, 2, 2-4 Hydrogen octyl trichloro pentane. Procedure: Place the SU-8 model wafer with 2-3 drops of decylating agent in a petri dish and place in a vacuum desiccator and leave it under vacuum for at least 丨 hours The composition is in a ratio of 10:1 (curing agent) (PDMS: SYLGARD® 184 is available from Dow Corning. SYLGARD is a vinyl group (Part A) and a hydrogenated oxoalkyl group (Part B). Two-part resin system) The pDMS prepolymer was prepared. The PDMS mixture was then added to a SU, 8 master wafer placed in a polystyrene Petri dish. The PDMS was cured in 24 hours without heating. (Alternatively, PDMS can be cured at 65 ° C for approximately 1-2 hours; depending on the thickness of the PDMS layer). The PDMS model is stripped from the SU-8 matrix, And cut to the right size. Available in David C. Duffy, J. Cooper McDonald, Olivier JA Schueller, and George M_Whitesides, “Rapid Prototyping of Microfluidic Systems in Poly(dimethyl oxalate) Of

Microfluidic Systems in Poly(dimethylsiloxane))，，Anal. 43 200912307Microfluidic Systems in Poly(dimethylsiloxane),, Anal. 43 200912307

Chem· 70, 1998.第4974-4984頁中找到額外的資訊，r 用方式納入本文中。 ％ 將PDMS流動通道附接至基質 PDMS設備具有鑄造至底表面内的狹窄流動通道。 PDMS設備使其與基f接觸，由pDMS設備提供流動通 牆壁和”天花板”，並由基質提供”地板”。流動通道的横切面 為長方形，寬度從100變化至3〇〇微米，且長度從5〇 至100微米。 將DNA附接至PDMS/基質 流動設備和基質是經過清潔的，並可視需要脫氣。以 標準汽相矽烷化草案製備基質表面，使用正-辛基三氯矽 烷。然後以最少的壓力將PDMS設備附接至基質。在流動 通遏的一端’利用接管導入10微升DNA(在IX TE中），然 後通過流動通道抽出。允許該溶液維持5至1〇分鐘以使末 化-附接反應穩定化。然後以每秒大約丨〇〇微米之流速，經 由通道移除流體。或者，使用基質而沒有流動設備，依靠 k準勿子梳草案’藉此將設備浸入DNA的溶液中，在5到 1 〇分知讓末端-附接反應穩定化之後，以每秒2〇〇_3〇〇微米 之速度撤回流體。在兩種例證方法中，以乙醇溶液沖 洗終產物’並以純水沖洗數次。 認為前述的書面說明書足以使熟諳此藝者得以實行本 發明。本發明在範圍上並不受限於所提供之實施例，因為 企圖將實施例當作本發明之一觀點的單純解釋，而其他在 44 200912307 上相等的具體事實亦在本發明之範圍内。從前述的 說B月中’除了在本文中出示和描述的那些之外 各種修改，對熟請此藝者而言會是顯而易見的，且亦^ ^之中請㈣範圍的範圍内。本發明的優點和目標不— 疋被納入本發明的每個具體事實中。 所有在本文令揭示的參考文獻’肖以引用方式納入本 文中’特別是上文提及的教示。 / 【圖式簡單說明】 圖式僅作為解釋之用，並不是使本文&amp; 施所必需的。 $月貫 圖1顯示方法步驟的概論。 圖2顯示排列製程的概論。 圖3顯示使用較大附接區之排列製程的概論。 圖4顯示基質附接區。 圖5顯示在製程中使用經標示和未經標示之腦。 圖6顯示重疊的螢光顯微照片(由 顯示寬度約6。微米)，顯示沉積在表面之平 又DNA分子0以YOYO-1染色DNA分子。77 圖7顯示如在圖6中所示之相同 但具有經調整之焦點面。注意到現在在置， 沉降在窗口上。 …點中有一些分子 圖8顯示在基質上相同位置 著螢光顯微分析顯# &lt; * 衫像。左圖顯示藉 …口結構的外形，帶有經排列但在 45 200912307 焦點範圍外的ADNA分子。右圖顯示如何改變爆點面，允 許分子在經扭曲窗口的頂部清晰地顯影。 圖9顯示拍攝基質的影像；不同的位置顯示又DNA分 子的沉降。顯然是由在焦點範圍外的DNA分子造成窗口扭 曲。 圖ίο和11顯示具有使用本發明方法製備之200奈米 #距的DNA分子之基質的TEM影像。 圖12顯示本發明之具體事實，其中在附接區排列該分 子。 圖13顯示本發明之具體事實，其中使用嚮導分子，以 產生分析物分子（欲分析之核酸分子）的間隔。 圖14顯示梯狀-構形DNA的TEM影像。以溴原子在兩 股上標不雙股DNA。每個平行線是兩個雙股之一。在暗線 之間的間隔輪流為1.5奈米和2奈米。2奈米距離是在相同 分子兩股的標記之間的距離。15奈米距離是在相鄰分子之 相鄰股的標記之間的間隔。整個分子齒距為大約3 ·5奈米。 【主要元件符號說明】 無 46 200912307 序列表 &lt;110&gt;ZS遺傳公司 &lt;120&gt;核酸分子之高密度分子排列 &lt;130&gt; Z0108.70004WO00 &lt;140&gt;未知 &lt;141&gt;附此 &lt;150&gt; US 60/937,066 &lt;151&gt; 2007-06-25 &lt;160&gt;3 &lt;170&gt; Patentln 3.3 版 &lt;210&gt; 1 &lt;211&gt;45 &lt;212&gt; DNA &lt;213&gt;人造的序列 &lt;220&gt; &lt;223&gt;合成的寡核苷酸 &lt;400&gt; 1 45 ggacggtctt cccagtcacg acactgtgta gaacgacggc ctgag &lt;210&gt;2 &lt;211&gt;41 &lt;212&gt; DNA &lt;213&gt;人造的序列 &lt;220&gt; &lt;223&gt;合成的寡核苷酸 &lt;400&gt; 2 ctcaggccgt cgttctacag agtgtcgtga ctgggaagc c 41 &lt;210 3 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt;人造的序列 &lt;220&gt; &lt;223&gt;合成的寡核苷酸 &lt;400&gt; 3 gtagaacgac ggcctgag 18 1Additional information is found in Chem. 70, 1998. Pages 4974-4984, which is incorporated herein by reference. % Attaching the PDMS Flow Channel to the Matrix The PDMS device has a narrow flow path cast into the bottom surface. The PDMS device is brought into contact with the base f, which provides a flow through wall and "ceiling" by the pDMS device and a "floor" from the substrate. The cross section of the flow channel is rectangular, with a width varying from 100 to 3 microns and a length from 5 100 to 100 microns. Attaching the DNA to the PDMS/matrix The flow device and substrate are cleaned and degassed as needed. The surface of the substrate was prepared using a standard vapor phase alkylation draft using n-octyltrichloromethane. The PDMS device is then attached to the substrate with minimal pressure. At the end of the flow repression, 10 μl of DNA (in IX TE) was introduced by a cannula, and then withdrawn through a flow channel. This solution was allowed to stand for 5 to 1 minute to stabilize the finalization-attachment reaction. The fluid is then removed through the passage at a flow rate of about 丨〇〇 microns per second. Alternatively, using a substrate without a flow device, relying on the k-pre-comb draft, thereby immersing the device in a solution of DNA, after 5 to 1 〇 knowing that the end-attachment reaction is stabilized, at 2 sec per second _3 〇〇 micron speed to withdraw the fluid. In both exemplary methods, the final product was washed with an ethanol solution and rinsed several times with pure water. It is believed that the foregoing written description is sufficient to enable those skilled in the art to practice the invention. The present invention is not limited by the scope of the present invention, and the specific embodiments of the invention are intended to be equivalent. From the foregoing, it is obvious that those who are present and described in this document, except those shown and described herein, will be apparent to those skilled in the art, and also within the scope of the scope of (4). The advantages and objectives of the present invention are not included in each particular aspect of the invention. All references cited in the present specification are incorporated herein by reference in their entirety, in particular the teachings mentioned above. / [Simple description of the schema] The schema is for illustrative purposes only and is not required for this article & $月图 Figure 1 shows an overview of the method steps. Figure 2 shows an overview of the alignment process. Figure 3 shows an overview of the alignment process using larger attachment areas. Figure 4 shows the matrix attachment zone. Figure 5 shows the use of labeled and unlabeled brains in the process. Figure 6 shows overlapping fluorescent micrographs (shown by a width of about 6. Micron) showing the deposition of DNA molecules on the surface and DNA molecules 0 staining the DNA molecules with YOYO-1. 77 Figure 7 shows the same as shown in Figure 6 but with an adjusted focal plane. Notice that it is now set and settled on the window. ... there are some molecules in the point. Figure 8 shows the same position on the substrate. Fluorescence microscopy shows the #&lt;* shirt image. The image on the left shows the shape of the borrowing structure with ADNA molecules aligned but outside the focus of 45 200912307. The image to the right shows how to change the burst surface, allowing the molecules to be clearly developed at the top of the warped window. Figure 9 shows an image of the photographing substrate; different locations show the sedimentation of DNA molecules. It is apparent that the window is twisted by DNA molecules outside the focus range. Figures ίο and 11 show TEM images of a substrate having a 200 nm DNA molecule prepared using the method of the present invention. Figure 12 shows the specific fact of the present invention in which the molecules are arranged in the attachment area. Figure 13 shows a specific fact of the present invention in which a guide molecule is used to generate an interval of an analyte molecule (a nucleic acid molecule to be analyzed). Figure 14 shows a TEM image of ladder-configuration DNA. The double-stranded DNA is superimposed on the two strands with a bromine atom. Each parallel line is one of two double strands. The spacing between the dark lines alternates between 1.5 nm and 2 nm. The 2 nm distance is the distance between the two strands of the same molecule. The 15 nm distance is the interval between the marks of adjacent strands of adjacent molecules. The entire molecular pitch is approximately 3-5 nm. [Explanation of main component symbols] None 46 200912307 Sequence Listing &lt;110&gt;ZS Genetics &lt;120&gt; High-density molecular arrangement of nucleic acid molecules &lt;130&gt; Z0108.70004WO00 &lt;140&gt;Unknown &lt;141&gt; Attached &lt;150&gt US 60/937,066 &lt;151&gt; 2007-06-25 &lt;160&gt;3 &lt;170&gt; Patentln version 3.3 &lt;210&gt; 1 &lt;211&gt;45 &lt;212&gt; DNA &lt;213&gt; artificial sequence&lt;220&gt;&lt;223&gt; Synthetic oligonucleotide &lt;400&gt; 1 45 ggacggtctt cccagtcacg acactgtgta gaacgacggc ctgag &lt;210&gt;2 &lt;211&gt;41 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;;223&gt; Synthetic oligonucleotide &lt;400&gt; 2 ctcaggccgt cgttctacag agtgtcgtga ctgggaagc c 41 &lt;210 3 &lt;211&gt; 18 &lt;212&gt; DNA &lt;213&gt; artificial sequence &lt;220&gt;&lt;223&gt; Oligonucleotide &lt;400&gt; 3 gtagaacgac ggcctgag 18 1

Claims (1)

200912307 十、申請專利範圍： 1 ·—種產製高密度間隔之核酸單層的方法，該方法包 括： 進行第一個附接步驟’以附接第一個核酸分子族群之 核酸分子的一端到基質的第一群接觸點，其中該第一群接 觸點是位在基質之表面的附接區中，並 使附接在該第—群接觸點之核酸分子排列，以產生第 一組經排列之核酸分子，其實質上是平行的，並以低於i 微米之間隔隔開， 藉以使該第一組經排列之核酸分子與該基質之表面結 口，藉此在该基質之表面上產生高密度間隔之核酸分子單 層，其包括該第一組經排列之核酸分子。 2·如申請專利範圍第1項之方法，更包括： ♦進行第二個附接步驟，以附接第二個核酸分子族群到 在第二群接觸點的附接區，並 二群接觸點之核酸分子排列，以產生第 分子’其實質上是平行的，並以低於丄 使附接至該第二群 I 一組經排列之核酸分子 微米之間隔隔開， 藉以使該第二組經排列之核酸分子與該基質200912307 X. Patent Application Range: 1 - A method for producing a high density spacer nucleic acid monolayer, the method comprising: performing a first attachment step 'to attach one end of a nucleic acid molecule of the first nucleic acid molecule group to a first group of contact points of the substrate, wherein the first group of contact points are in an attachment region of the surface of the substrate, and the nucleic acid molecules attached to the first group contact point are aligned to produce a first set of aligned Nucleic acid molecules, which are substantially parallel and spaced at intervals less than i microns, whereby the first set of aligned nucleic acid molecules are associated with the surface of the substrate, thereby producing on the surface of the substrate A high density interstitial nucleic acid molecule monolayer comprising the first set of aligned nucleic acid molecules. 2. The method of claim 1, further comprising: ♦ performing a second attachment step to attach the second nucleic acid molecule population to the attachment zone at the second group of contact points, and the two group of contact points Nucleic acid molecules are arranged to produce a first molecule 'which is substantially parallel and separated by an interval below the 丄 to a micron of a group of aligned nucleic acid molecules attached to the second group I, thereby making the second group Aligned nucleic acid molecule with the substrate 包括該第一和第二組經排列之核酸分子。 該基質之表面結 核酸分子單The first and second sets of aligned nucleic acid molecules are included. The surface of the matrix is a nucleic acid molecule 群接觸點。 1項之方法，其中該第—個附接包 第一個核酸分子族群附接至第— 200912307 4.如申明專利範圍第2項之方法其中該第二個附接包 括經由第一群連接子使該第二個核酸分子族群附接至第二 群接觸點。 5·如申請專利範圍第3項之方法’其中該第—個連接子 為寡核苦酸。 6.如中請專利範圍第4項之方法，其中該第二個連接子 為养核苦酸。 7·如申請專利！i圍第3項之方法，其中該第—個連接子 包括正-辛基基團。 其中該第二個連接子 8.如申請專利範圍第4項之方法 包括正-辛基基團。 9,如申請專利範圍第“戈2項之方法，其中該第—或第 二個附接步驟包括藉著在該基質之表面和該核酸分子之間 形成一或多個共價鍵，使該核酸分子附接。Group contact points. The method of claim 1, wherein the first nucleic acid molecule population of the first attachment package is attached to the first - 200912307. 4. The method of claim 2, wherein the second attachment comprises via the first group of connectors The second population of nucleic acid molecules is attached to a second group of contacts. 5. The method of claim 3, wherein the first linker is an oligonucleotide. 6. The method of claim 4, wherein the second linker is a nutrient. 7·If you apply for a patent! The method of item 3, wherein the first linker comprises a n-octyl group. Wherein the second linker 8. The method of claim 4 includes a n-octyl group. 9. The method of claim 2, wherein the first or second attachment step comprises: by forming one or more covalent bonds between the surface of the substrate and the nucleic acid molecule Nucleic acid molecule attachment. 10·如申請專利範圍第…項之方法，其中該第—或 第二個附接步驟包括υν·交聯、在磷酸基團和經官能化的表 面之間產生離子吸引力、使用經修飾之DNA以結合^ 質表面修飾，及/或使用陽離子表面處理。 -基 11.如申請專利範圍第m項中任一項之方法，其中 ^ 列包括指引流體流動跨越該基質。 12·如申請專利範圍第1至8項中任一項 該排列包括對該核酸分子施加電場。 ’、中 13.如申請專利範圍第…項中任一 該排列包括對該核酸分子施加磁場。 /、中 2 200912307 Ι4·如申凊專利範圍第丨 斗杜併a μ 8項中任一項之方法，其中 該基質包括排列區。 15. 如申請專利範圍第M項 &lt;方法’其中該排列致使該 核S文分子在該基質之排列區上排列。 項中任一項之方法，其中 16. 如申請專利範圍第ί至 該基質包括薄膜。 17.如申凊專利範圍第μ頊之古ί ^ ^ 項之方去，其中該薄膜為奈米 級的膜。10. The method of claim 2, wherein the first or second attachment step comprises υν·crosslinking, generating an ionic attraction between the phosphate group and the functionalized surface, using a modified The DNA is modified with a binding surface and/or treated with a cationic surface. The method of any one of the preceding claims, wherein the column comprises directing fluid flow across the substrate. 12. The method of any one of claims 1 to 8 wherein the arrangement comprises applying an electric field to the nucleic acid molecule. ', 13. The application of any of the scope of the patent application includes the application of a magnetic field to the nucleic acid molecule. The method of any of the preceding claims, wherein the substrate comprises an alignment zone. 15. The scope of the patent application, item M &lt;method&apos; wherein the arrangement causes the core S molecules to be aligned on the aligned regions of the substrate. The method of any one of the preceding claims, wherein, as claimed in the patent application, the substrate comprises a film. 17. For example, the application of the patent range 第μ顼古ί ^ ^, where the film is a nano-scale film. ί 18. 如申請專利範圍第！至8項中任—項之方法，其中 該經排列之核酸分子實質上是直線的。 19. 如申請專利範圍第…項中任一項之方法，其中 該基質包括石墨表面。 ' 20. 如申請專利範圍第丨至8項中任一項之方法，其中 §亥經排列之分子為梯狀構形。 21. 如申請專利範圍第1至8項中任一項之方法，其中 该第一個核酸分子族群是經標示之核酸分子。 22. 如申請專利範圍第ί至8項中任一項之方法，其中 §亥第一個核酸分子族群是未經標示之核酸分子。 23·如申請專利範圍第2炱8項中任一項之方法，其中 該第二個核酸分子族群是經標示之核酸分子。 24. 如申請專利範圍第2直8項中任一項之方法，其中 該第二個核酸分子族群是未經梯示之核酸分子。 25. 如申請專利範圍第21頊之方法，其中該標記是可使 用牙透式電子顯微鏡（ΤΕΜ)檢測的。 3 200912307 26. 如申請專利範圍第25項之方法其中該標記包括 具有可與在得自TEM之影像上的背景雜訊對比來檢測之 核電荷的原子。 27. 如申請專利範圍第26項之方法，其中該標記包括 一至五個非-螢光原子。 28·如申請專利範圍第1至8項中任一項之方法，其 中該核酸分子之長度為至少1 00個鹼基對。 29·如申請專利範圍第1至8項中任一項之方法，其 中該核酸分子之長度為至少1 〇〇〇個鹼基對。 30·如申請專利範圍第1至8項中任一項之方法，其 中該核酸分子之長度為至少1〇,〇〇〇個鹼基對。 3 1 _如申請專利範圍第1至8項中任一項之方法，其 中該核酸分子之長度為至少20,000個鹼基對。 32.如申請專利範圍第1至8項中任一項之方法，其 中該經排列之分子與該基質表面的結合是非-共價的。 3 3.如申請專利範圍第1至 8項中任一項之方法，其 中該經排列之分子與該基質表面的結合是可逆的。 34.如申請專利範圍第1至8項中任一項之方法，其 中與該表面之結合包括使定向之流體流動減緩及/或停止、 改隻&quot;IL體及/或環境之溫度、在使流體流動停止之後以定向 之方式使核酸分子脫水，及/或在移除流體之前或之後，加 入促進與表面之結合的試劑及/或活化劑。 3 5 .如申請專利範圍第1至8項中任一項之方法，其 中該與表面之結合包括藉著在該基質表面和該核酸分子之 4 200912307 間形成一或多個共價键 . 、1貝鍵，使核酸分子附接。 3 6.如申請專利銘阁&amp; 摩已圍第1至8項中任一項之方法，其 中該與表面之結合台虹上 匕栝UV-父聯、在磷酸基團和經官能化 的表面之間產生離子丨士 卞吸引力、使用經修飾之DNA以結合 其他基質表面修飾，及/式描田哄私，士 及’或使用％離子表面處理。 3 7.如申請專利範圊筮κ ts 乾圍弟36項之方法，其中該連接子和 表面修飾是在該基質上相同的區域。 3 8.如申請專利筋圍链$ 寸π軏圍第1至8項中任一項之方法，其 中該與表面之結合包括錄i社人^ 匕括I由結合對，使該核酸分子附接至 該表面。 其 39.如申請專利範圍帛i至8項中任一項之方法 中至少一部分的基質表面是疏水性的。 其 4〇.如申請專利範圍第1至8項中任一項之方法 中至少-部分的基質表面是經官能化而成為疏水性的。 其 41·如申請專利範圍第丨至8項中任—項之方法 中該高密度間隔之核酸分子單層是部分單層。 其 42. 如申請專利範圍第丨至8項中任一項之方法且 中至少25%的經排列之核酸分子以低於1〇〇 / : 、不I間隔 開。 43. 如申請專利範圍第42項之方法，盆中 /、甲至少5〇%的 經排列之核酸分子以低於10 0奈米之間隔隔開。 44. 如申請專利範圍第1至8項中任— 、 貝之方法，其 中至少25°/〇的經排列之核酸分子以低於1〇太 ' '丁、未之間隔隔 200912307 5 0%的 45. 如申請專利範圍第44項之方法，复Λ 丹肀至少 經排列之核酸分子以低於1 0奈米之間隔隔開。 項之方法’其 46. 如申請專利範圍第1至 8項中任_ 中至少25%的經排列之核酸分子是相碰觸的 5 0%的 47·如申請專利範圍第46項之方法，其中至小 經排列之核酸分子是相碰_觸的。 48. 如申請專利範圍第1至 8項中任— 貝之方法，立 中至少2 5 %的經排列之核酸分子是以使經押+1 知不和未經標示 之核酸分子交替的方式來排列。 49. 如申請專利範圍第48項之方法，发φ 5 , ”中至少5〇%的 經排列之核酸分子是以使經標示和未經標示 &amp;核酸分子交 替的方式來排列。 50. 如申請專利範圍第丨至8項中任—箱 貝之方法，其 中將一或多個磁珠附接在該核酸分子上，且复 Τ稽者施加 磁力來排列該核酸分子。ί 18. If you apply for a patent scope! The method of any of the items 8, wherein the aligned nucleic acid molecules are substantially linear. 19. The method of any one of the preceding claims, wherein the substrate comprises a graphite surface. 20. The method of any one of claims 00 to 8, wherein the molecules arranged in the wales are ladder-shaped. The method of any one of claims 1 to 8, wherein the first nucleic acid molecule population is a labeled nucleic acid molecule. 22. The method of any one of claims 00 to 8, wherein the first nucleic acid molecule population is an unlabeled nucleic acid molecule. The method of any one of claims 2 to 8, wherein the second nucleic acid molecule population is a labeled nucleic acid molecule. 24. The method of any one of claims 2 to 8, wherein the second population of nucleic acid molecules is a nucleic acid molecule that is not shown. 25. The method of claim 21, wherein the label is detectable using a dental electron microscope (ΤΕΜ). The method of claim 25, wherein the indicia comprises an atom having a nuclear charge detectable in contrast to background noise on an image obtained from the TEM. 27. The method of claim 26, wherein the label comprises from one to five non-fluorescent atoms. The method of any one of claims 1 to 8, wherein the nucleic acid molecule is at least 100 base pairs in length. The method of any one of claims 1 to 8, wherein the nucleic acid molecule is at least 1 base pair in length. The method of any one of claims 1 to 8, wherein the nucleic acid molecule is at least 1 〇 in length, 〇〇〇 one base pair. The method of any one of claims 1 to 8, wherein the nucleic acid molecule is at least 20,000 base pairs in length. The method of any one of claims 1 to 8, wherein the alignment of the aligned molecules with the surface of the substrate is non-covalent. The method of any one of claims 1 to 8, wherein the alignment of the aligned molecules with the surface of the substrate is reversible. The method of any one of claims 1 to 8, wherein the combination with the surface comprises slowing and/or stopping the directed fluid flow, changing only the temperature of the IL body and/or the environment, The nucleic acid molecules are dehydrated in an oriented manner after the fluid flow is stopped, and/or reagents and/or activators that promote binding to the surface are added before or after the fluid is removed. The method of any one of claims 1 to 8, wherein the binding to the surface comprises forming one or more covalent bonds between the surface of the substrate and the nucleic acid molecule 4 200912307. 1 shell bond to attach nucleic acid molecules. 3 6. The method of claiming any one of items 1 to 8 in which the combination of the surface and the surface is combined with a UV-parent, a phosphate group and a functionalized The interaction between the surfaces is ionic, the use of modified DNA to bind other substrate surface modifications, and the use of % ion surface treatment. 3 7. A method as claimed in claim 36, wherein the linker and the surface modification are the same region on the substrate. 3 8. The method of claiming any one of items 1 to 8 of the patent rib chain, wherein the combination with the surface comprises recording the binding of the nucleic acid molecule. Connect to the surface. 39. The surface of the substrate of at least a portion of the method of any one of claims 帛i to 8 is hydrophobic. The method of any one of the methods of any one of claims 1 to 8 wherein at least a portion of the surface of the substrate is functionalized to become hydrophobic. 41. The method of claim 1, wherein the high density interstitial nucleic acid molecule monolayer is a partial monolayer. 42. The method of any one of claims 00 to 8 wherein at least 25% of the aligned nucleic acid molecules are spaced apart by less than 1 〇〇 / :, not 1. 43. The method of claim 42, wherein at least 5% of the aligned nucleic acid molecules in the pot are separated by less than 100 nanometers. 44. The method of claim 1, wherein at least 25°/〇 of the aligned nucleic acid molecules are less than 1〇太′′, and the interval is not separated by 200912307 50%. 45. The method of claim 44, wherein at least the aligned nucleic acid molecules are separated by less than 10 nanometers. The method of the 'subjects'. At least 25% of the aligned nucleic acid molecules in any of the claims 1 to 8 are 50% of the touched 47. The method of claim 46, The nucleic acid molecules arranged to the small ones are in contact with each other. 48. As in the method of applying for the patents in items 1 to 8, the method of arranging at least 25 % of the aligned nucleic acid molecules is to alternate between the +1 and the unlabeled nucleic acid molecules. arrangement. 49. As claimed in claim 48, at least 5% of the aligned nucleic acid molecules in the φ 5 , ” are arranged in such a way that the labeled and unlabeled &amp; nucleic acid molecules alternate. The method of claim </ RTI> wherein the one or more magnetic beads are attached to the nucleic acid molecule, and the repeller applies a magnetic force to align the nucleic acid molecule. 51. —種高密度間隔之核酸分子單層，其包括 一基質，和 附接在該基質上的經排列之核酸分子，以低於丨微米 之間隔隔開。 52.如申請專利範圍第51項之高密度間隔之核酸分子 單層，其中該單層包括第一個核酸分子族群，其附接在該 基質的第一群接觸點上。 53·如申請專利範圍第52項之高密度間隔之核酸分子 單層’其中該第-群接觸點係位在該基質之表面上的附接 200912307 區0 54. 如申請專利範圍第5丨項之高密度間隔之核酸分子 單層，其中该經排列之核酸分子包括附接在該基質表面之 多個點的第一組經排列之核酸分子。 55. 如申請專利範圍第5 I項之高密度間隔之核酸分子 單層，其中該單層包括第二個核酸分子族群，其附接在該 基質的第二群接觸點上。 56. 如申請專利範圍第55項之高密度間隔之核酸分子 C 單層，其中3亥第二群接觸點係位在該基質之表面上的附接 區。 57·如申請專利範圍第51至56項中任一項之高密度間 隔之核酸分子單層，其中該基質具有排列區。 58_如申請專利範圍第51至56項中任一項之高密度間 隔之核酸分子單層，其中該基質具有石墨表面。 59.如申請專利範圍第51至S6項中任一項之高密度 間隔之核酸分子單層，其中該經排列之核酸分子包括附接 I : 在該基質表面之多個點的第二組經排列之核酸分子。 60·如申請專利範圍第51至56項中任一項之高密度 間隔之核酸分子單層，其中該經排列之核酸分子實質上是 直線的。 61.如申請專利範圍第60項之高密度間隔之核酸分子 單層’其中該經排列之核酸分子為梯狀構形。 62·如申請專利範圍第51至56項中任一項之高密度 間隔之核酸分子單層，其中該經排列之核酸分子是經標示 7 200912307 之核酸分子。 63. 如申明專利範圍第5丨至56項中任一項之高密度 間隔之核酸分子單層，#中該核酸分子是經標示和未經標 示之核酸分子的混合物。 64. 如申清專利範圍第62項之高密度間隔之核酸分子 單層其巾4 &amp; §己是可使用穿透式電子顯微鏡（tem)檢測 的。51. A high density interstitial nucleic acid molecule monolayer comprising a matrix, and aligned nucleic acid molecules attached to the substrate, separated by an interval of less than one micron. 52. A high density interstitial nucleic acid molecule monolayer according to claim 51, wherein the monolayer comprises a first nucleic acid molecule population attached to a first group of contact points of the matrix. 53. A high density interstitial nucleic acid molecule monolayer as described in claim 52, wherein the first group contact point is attached to the surface of the substrate. 200912307 Region 0 54. A high density interstitial nucleic acid molecule monolayer, wherein the aligned nucleic acid molecule comprises a first set of aligned nucleic acid molecules attached to a plurality of points on the surface of the substrate. 55. A single layer of a high density spacer nucleic acid molecule according to claim 5, wherein the monolayer comprises a second population of nucleic acid molecules attached to a second group of contacts of the matrix. 56. A high density interstitial nucleic acid molecule C monolayer according to claim 55, wherein the 3H second group of contact points are attached to an attachment region on the surface of the substrate. The high density substrate nucleic acid molecule monolayer according to any one of claims 51 to 56, wherein the substrate has an alignment region. The nucleic acid molecule monolayer of the high density interval of any one of claims 51 to 56, wherein the substrate has a graphite surface. The high density interstitial nucleic acid molecule monolayer according to any one of claims 51 to 6, wherein the aligned nucleic acid molecule comprises an attachment I: a second set of points at a plurality of points on the surface of the substrate Arranged nucleic acid molecules. The high density interstitial nucleic acid molecule monolayer of any one of claims 51 to 56, wherein the aligned nucleic acid molecules are substantially linear. 61. A high density interstitial nucleic acid molecule monolayer&apos; as claimed in claim 60, wherein the aligned nucleic acid molecule is in a ladder configuration. The high density interstitial nucleic acid molecule monolayer according to any one of claims 51 to 56, wherein the aligned nucleic acid molecule is a nucleic acid molecule designated 7 200912307. 63. A high density interstitial nucleic acid molecule monolayer according to any one of claims 5 to 56, wherein the nucleic acid molecule is a mixture of labeled and unlabeled nucleic acid molecules. 64. A high-density spacer nucleic acid molecule such as the patent scope 62 is a single layer of the towel 4 &amp; § can be detected using a transmission electron microscope (TEM). 65_如申請專利範圍第“項之高密度間隔之核酸分子 早層，其中該標記包括具有可與在得自ΤΕΜ之影像上的 背景雜訊對比來檢測之分子量的原子。 6 6.如申請專利範圍第 單層，其中該標記包括一至 67.如申請專利範圍第51至56項 間隔之核酸分子單層，其中該核酸分子 個驗基對。 65項之高密度間隔之核酸分 五個非-螢光原子。 子 中任一項之高密度 之長度為至少100 68.如申請專利範 間隔之核酸分子單層 個鹼基對。 圍第51至％項中任一項之高密度 ’其中該核酸分子之長度為至少1〇〇〇 69.如申請專利範圊坌 』乾圍第51至56項中任一項之高密度 間隔之核酸分子單層，甘士 # ;π 層其中該核酸分子之長度為至少10,000 個鹼基對。 項中任一項之高密度 之長度為至少20,〇〇〇 70·如申凊專利範圍第51至56 間隔之核酸分子單層，其中該核酸分子 個鹼基對。 8 200912307 71. 如申請專利範圍第51至％項中任一項之高密度 間隔之核酸分子單層，λ中藉著連接子將該核酸分子附接 至該基質之表面。 72. 如申請專利範圍第51至％項中任一項之高密度 間隔之核酸分子單層，#中藉著在該基質表面和該核酸分 子之間的一或多個共價鍵，將該核酸分子附接至該基質之 表面。 73. 如申請專利範圍第51至56項中任一項之高密度 間隔之核酸分子單層，其中至少一部分的基質表面為疏水 性的。 74·如申請專利範圍第73項之高密度間隔之核酸分子 單層’其中該基質具有薄膜。 75.如申請專利範圍第74項之高密度間隔之核酸分子 單層’其中該薄膜為奈米級的膜。 76·如申凊專利範圍第5 1至56項中任一項之高密度 間隔之核酸分子單層，其中至少一部分的基質表面是經官 能化而成為疏水性的。 77·如申請專利範圍第51至56項中任一項之高密度 間隔之核酸分子單層，其中該單層為部分單層。 78·如申請專利範圍第51至56項中任一項之高密度 間隔之核酸分子單層’其中至少25%的經排列之核酸分子 以低於1 〇〇奈米之間隔隔開。 79·如申請專利範圍第78項之高密度間隔之核酸分子 單声，甘士 曰’八中至少50%的經排列之核酸分子以低於100奈米 9 200912307 之間隔隔開。 80.如申請專利範圍第51至56項中任一項之高密度 間隔之核酸分子單層，其中至少25%的經排列之核酸分子 以低於10奈米之間隔隔開。 8 1.如申請專利範圍第80項之高密度間隔之核酸分子 單層’其中至少50%的經排列之核酸分子以低於1 〇奈米 之間隔隔開。 82. 如申請專利範圍第5 1至56項中任一項之高密度 間隔之核酸分子單層，其中至少25%的經排列之核酸分子 是相碰觸的。 83. 如申請專利範圍第82項之高密度間隔之核酸分子 單層’其中至少50%的經排列之核酸分子是相碰觸的。 84.如申請專利範圍第51至56項中任一項之高密度 間隔之核酸分子單層，其中至少25%的經排列之核酸分子 是以使經標示和未經標示之核酸分子交替的方式來排列。 d〇 85·如申凊專利範圍第83項之高密度間隔之核酸分子 單層’其中至纟50%的經排列之核酸分子是以使經標示和 未經標示之核酸分子交替的方式來排列。65. An early layer of a nucleic acid molecule of high density spacing as set forth in the scope of the patent application, wherein the label comprises an atom having a molecular weight detectable in comparison to background noise on an image obtained from sputum. 6 6. If applied The patented range is a single layer, wherein the label comprises from one to 67. A single layer of a nucleic acid molecule at intervals of 51 to 56 of the patent application, wherein the nucleic acid molecule has a pair of nucleotides. The nucleic acid of the high density interval of 65 is divided into five non- - a fluorescent atom. The high density of any one of the sub-lengths is at least 100 68. A single layer of base pairs of nucleic acid molecules as claimed in the patent application. High density of any one of items 51 to % The nucleic acid molecule has a length of at least 1〇〇〇69. The nucleic acid molecule monolayer of the high-density spacer according to any one of the items 51 to 56 of the patent specification, Gans #; π layer, wherein the nucleic acid The length of the molecule is at least 10,000 base pairs. The high density of any one of the items is at least 20, 〇〇〇 70 · a single layer of a nucleic acid molecule at intervals 51 to 56 of the claimed patent range, wherein the nucleic acid molecule Base pairs. A high-density spacer nucleic acid molecule monolayer according to any one of claims 51 to 10, wherein the nucleic acid molecule is attached to the surface of the substrate by a linker in λ. 72. A single layer of a high density spacer nucleic acid molecule according to any one of items 51 to 10, wherein the nucleic acid molecule is attached to the nucleic acid molecule by one or more covalent bonds between the surface of the substrate and the nucleic acid molecule The high density interstitial nucleic acid molecule monolayer according to any one of claims 51 to 56, wherein at least a portion of the substrate surface is hydrophobic. 74. A high-density spacer nucleic acid molecule monolayer in which the matrix has a film. 75. A high-density spacer nucleic acid molecule monolayer according to claim 74, wherein the film is a nano-scale film. The high density interstitial nucleic acid molecule monolayer of any one of clauses 51 to 56, wherein at least a portion of the substrate surface is functionalized to become hydrophobic. 77. In the scope of claims 51 to 56 Either a high-density spacer nucleic acid molecule monolayer, wherein the monolayer is a partial monolayer. 78. The high-density spacer nucleic acid molecule monolayer of any one of claims 51 to 56, wherein at least 25% of the Arranged nucleic acid molecules are separated by less than 1 〇〇 nanometer. 79. High-density spacer nucleic acid molecules as claimed in Article 78 of the patent application, at least 50% of aligned nucleic acids in Gans 曰 'eight The molecules are separated by a spacing of less than 100 nm 9 200912307. 80. A high density interstitial nucleic acid molecule monolayer according to any one of claims 51 to 56, wherein at least 25% of the aligned nucleic acid molecules are Separated by intervals below 10 nm. 8 1. A high density interstitial nucleic acid molecule as claimed in claim 80, wherein at least 50% of the aligned nucleic acid molecules are separated by less than 1 nanometer. 82. A high density interstitial nucleic acid molecule monolayer according to any one of clauses 51 to 56, wherein at least 25% of the aligned nucleic acid molecules are in contact. 83. A high density interstitial nucleic acid molecule monolayer&apos; as claimed in claim 82, wherein at least 50% of the aligned nucleic acid molecules are in contact. 84. A high density interstitial nucleic acid molecule monolayer according to any one of claims 51 to 56, wherein at least 25% of the aligned nucleic acid molecules are in a manner that alternates between labeled and unlabeled nucleic acid molecules. To arrange. D〇85· A high-density spacer nucleic acid molecule monolayer of claim 83, wherein up to 50% of the aligned nucleic acid molecules are arranged in such a way that alternating labeled and unlabeled nucleic acid molecules . 獲得經標示之核酸分子族群，Obtaining a labeled nucleic acid molecule population, 4，作為如根據申請專利 或第二個核酸分子族群， 200912307 產製高密度間隔之核酸分子單層，成 使用粒子束判讀該單層之核酸分子的序列。 88.如申請專利範圍第87項之方法’更包括藉著下列 步驟產製經標示之核酸分子族群 從°式樣中獲得核酸分子族群，姐 以—或多個標記標示該核酸分子之核苷酸，該標記是 彳灸用蜜1 、悉 逐式電子顯微鏡（ΤΕΜ)檢測的，以產製經標示之 核酸分子族群。 89·如申晴專利範圍第23項之方法，其中該標記是可 使用牙透式電子顯微鏡（ΤΕΜ)檢測的。 9〇·如申請專利範圍第89項之方法，其中該標記包括 有可與在彳于自ΤΕΜ之影像上的背景雜訊對比來檢測之 核電荷的原子。 91·如申請專利範圍帛9〇項之方法，其中該標記包括 一至五個非-螢光原子。 92.如申請專利範圍第63項之高密度間隔之核酸分子 單層，戈* Φ与Γ # _χ Ώ 、Μ不5疋可使用穿透式電子顯微鏡（ΤΕΜ)檢測 單層 背景 ，如申1專利範圍第92項之高密度間隔之核酸分子 “中該‘ 3己包括具有可與在得自τεμ《影像上的 雜訊對比來檢測之分子量的原子。 單層 94.如申請專利範圍第 其中该標記包括一至 93項之高密度間隔之核酸分子 五個非-螢光原子。 114. As a single layer of a nucleic acid molecule having a high density of intervals, according to the patent application or the second nucleic acid molecule group, 200912307, the sequence of the nucleic acid molecule of the single layer is read using a particle beam. 88. The method of claim 87, further comprising the step of producing a population of nucleic acid molecules from the model by the following steps, and identifying the nucleotides of the nucleic acid molecule with or The marker is acupuncture and moxibustion, and is detected by an electron microscope (ΤΕΜ) to produce a labeled nucleic acid molecule group. 89. The method of claim 23, wherein the label is detectable using a dental electron microscope (ΤΕΜ). 9. The method of claim 89, wherein the mark comprises an atom having a nuclear charge detectable against background noise on an image that is self-defeating. 91. The method of claim 9, wherein the label comprises from one to five non-fluorescent atoms. 92. If the high-density spacer nucleic acid molecule monolayer of claim 63 is applied, Ge*Φ and Γ# _χ Μ, Μ5疋 can be used to detect a single layer background using a transmission electron microscope (ΤΕΜ), such as Shen 1 The high-density spacer nucleic acid molecule of the 92nd patent of the patent scope "includes an atom having a molecular weight detectable in comparison with the noise obtained from the τεμ" image. Single layer 94. The label comprises from one to 93 high density interstitial nucleic acid molecules of five non-fluorescent atoms.