JPH0275958A - Detection of dna - Google Patents
Detection of dnaInfo
- Publication number
- JPH0275958A JPH0275958A JP22757788A JP22757788A JPH0275958A JP H0275958 A JPH0275958 A JP H0275958A JP 22757788 A JP22757788 A JP 22757788A JP 22757788 A JP22757788 A JP 22757788A JP H0275958 A JPH0275958 A JP H0275958A
- Authority
- JP
- Japan
- Prior art keywords
- dna
- probe
- fluorescent
- strands
- formation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title abstract description 10
- 239000000523 sample Substances 0.000 claims abstract description 41
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 17
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 12
- 125000003277 amino group Chemical group 0.000 claims abstract description 7
- 238000004458 analytical method Methods 0.000 claims abstract description 6
- 239000003298 DNA probe Substances 0.000 claims abstract description 5
- 238000006073 displacement reaction Methods 0.000 claims abstract description 4
- ZBKFYXZXZJPWNQ-UHFFFAOYSA-N isothiocyanate group Chemical group [N-]=C=S ZBKFYXZXZJPWNQ-UHFFFAOYSA-N 0.000 claims abstract description 4
- 108020004414 DNA Proteins 0.000 claims description 59
- 238000000034 method Methods 0.000 claims description 14
- 102000053602 DNA Human genes 0.000 claims description 6
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 4
- 238000002875 fluorescence polarization Methods 0.000 claims description 4
- 238000003745 diagnosis Methods 0.000 abstract description 7
- 201000010099 disease Diseases 0.000 abstract description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 5
- 230000001268 conjugating effect Effects 0.000 abstract 2
- 108020003215 DNA Probes Proteins 0.000 abstract 1
- 238000011321 prophylaxis Methods 0.000 abstract 1
- 238000011282 treatment Methods 0.000 abstract 1
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- 108090000623 proteins and genes Proteins 0.000 description 9
- 230000000295 complement effect Effects 0.000 description 5
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- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 3
- 208000026350 Inborn Genetic disease Diseases 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
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- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
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- 230000005653 Brownian motion process Effects 0.000 description 2
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000002105 Southern blotting Methods 0.000 description 2
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- 102100033215 DNA nucleotidylexotransferase Human genes 0.000 description 1
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- WUBNGZXADUXZOF-PBWSWVRCSA-N [[(2r,3s,4r,5s)-5-amino-5-[6-amino-8-(6-aminohexyl)purin-9-yl]-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound NCCCCCCC1=NC2=C(N)N=CN=C2N1[C@]1(N)O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O WUBNGZXADUXZOF-PBWSWVRCSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
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- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
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Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は、ＤＮＡの検出法に関するものである。[Detailed description of the invention] (Industrial application field) The present invention relates to a method for detecting DNA.
更に詳細には１本発明は、医療、薬品、食品等の分野に
おいて、遺伝子あるいは感染症を誘発するウィルス等の
特定の塩基配列を有するＤＮＡの検出法に関する。More specifically, the present invention relates to a method for detecting DNA having a specific base sequence, such as a gene or a virus that induces an infectious disease, in the fields of medicine, medicine, food, etc.
一般に、ＤＮＡの診断や変異ＤＮＡの検出にはプローブ
（探索子）が用いられ、プローブが結合するが、結合し
ないかによって、診断や検出の判断が行なわれている。Generally, probes (probes) are used for DNA diagnosis and detection of mutant DNA, and diagnosis and detection are determined depending on whether the probe binds or not.
本発明では蛍光色素で標識されたＤＮＡをプローブとし
て用いることによって、ＤＮＡと結合したかどうかをす
みやかに測定することができるようになるので、医療、
薬品、食品等バイオ関連技術におけるＤＮＡの診断や変
異ＤＮＡの検出に大きく貢献するものである。In the present invention, by using DNA labeled with a fluorescent dye as a probe, it is possible to quickly measure whether or not it has bound to DNA, so it can be used in medical,
This will greatly contribute to DNA diagnosis and detection of mutant DNA in bio-related technologies such as pharmaceuticals and foods.
（従来技術及び問題点）
遺伝子であるＤＮＡは、４種の核酸塩基が高度に配列し
た２重らせん構造を構成しており、この塩基配列には、
ホルモン、酵素等の蛋白質の組成。(Prior art and problems) DNA, which is a gene, has a double helix structure in which four types of nucleobases are highly arranged.
Composition of proteins such as hormones and enzymes.
構造を決定する重要な遺伝情報が組み込まれている。こ
のヒトの遺伝子は５〜１０万といわれるが、遺伝的ある
いは突然変異によって生じる異常や変化、あるいはウィ
ルスなどの外界から生体に侵入してくる外因性遺伝子に
よって生体機能のバランスが損なわれる。この遺伝子レ
ベルでの分析は、従来より臨床分野において染色体検査
として行われているが塩基配列として１００万塩基以上
のものしか検出できず、これらは転座や欠失といった染
色体の構造異常として検査しているにすぎない。It contains important genetic information that determines its structure. Humans are said to have 50,000 to 100,000 genes, but the balance of biological functions can be disrupted by abnormalities or changes caused by genetic or mutation, or by exogenous genes that invade the body from the outside world, such as viruses. This gene-level analysis has traditionally been carried out as a chromosome test in the clinical field, but it can only detect base sequences of 1 million bases or more, and these can only be detected as chromosomal structural abnormalities such as translocations and deletions. It's just that.
従って遺伝病等の遺伝子の変化、あるいは外因性遺伝子
に起因する疾患の診断にあたっては、臨床的な所見や生
化学的な検査法以外に直接ＤＮＡの変異を検出すること
ができれば非常に有力な手段となりうる。Therefore, in diagnosing genetic changes such as genetic diseases or diseases caused by exogenous genes, it would be an extremely effective method to directly detect DNA mutations in addition to clinical findings and biochemical testing methods. It can be.
最近では、比較的再現性のよいＤＮＡの操作技術が確認
され、またさまざまなりＮＡプローブの開発によってＤ
ＮＡを直接検出することが可能となり、ＤＮＡ診断とし
て臨床検査への応用が急速に注目されつつある。このＤ
ＮＡ診断は病気という表現型の変化が生じる前に病気の
診断や予測を行うことが可能であり、さらに、遺伝病の
出生前診断においては、羊水中の細胞ＤＮＡを検査する
ことによって胎児の遺伝病の判定が可能、すなわち、ど
の体細胞のＤＮＡでも検査対象とすることができる。Recently, DNA manipulation techniques with relatively high reproducibility have been confirmed, and various NA probes have been developed.
It has become possible to directly detect NA, and its application to clinical testing as DNA diagnosis is rapidly gaining attention. This D
NA diagnosis makes it possible to diagnose and predict diseases before phenotypic changes occur.Furthermore, in prenatal diagnosis of genetic diseases, it is possible to determine the genetic status of a fetus by testing cellular DNA in amniotic fluid. It is possible to determine the disease, that is, the DNA of any somatic cell can be tested.
現在ＤＮＡに関する検究は、 ＲＦＬＰ（１’ｌｅｓｔ
ｒｉｃｔｉｏｎＦｒａｇｍｅｎｔ Ｌｅｎｇｔｈ Ｐｏ
ｌｙｍｏｒｐｈｉｓｍ）連鎖解析を中心として活発に行
われ、特に約３０種の遺伝病の遺伝子の変異も報告され
ている。検出法としては適当な制限酵素で切断したＤＮ
Ａ断片をポリアクリルアミド、あるいはアガロースゲル
等を用いる電気泳動法によって分離、ニトロセルロース
膜などに固定化後、マーカーで標識されたプローブＤＮ
Ａとのハイブリダイゼーション（ＤＮＡの２本鎖形成）
により検出する、いわゆるサザンブロッティング法が主
流である。マーカーとしては従来より、放射性同位体（
ＲＩ）が用いられ高感度分析が行われてきたが、ＲＩが
高価であり、また取り扱いの危険性、半減期が短く安定
したプローブを得にくいなどの問題がある。このような
観点から非ＲＩ標識プローブの開発が盛んに行われてき
た。これらには、ビオチン−アビジン法、ハプテン標識
法、ラベザイムーＰＯＤ法、ケミプローブ法などがあげ
られるが。Currently, DNA tests are conducted using RFLP (1'rest
rictionFragment Length Po
lymorphism) linkage analysis has been actively conducted, and mutations in the genes of about 30 genetic diseases have been reported. As a detection method, DNA cut with an appropriate restriction enzyme is used.
After separating the A fragment by electrophoresis using polyacrylamide or agarose gel, etc. and immobilizing it on a nitrocellulose membrane, probe DNA labeled with a marker is obtained.
Hybridization with A (double strand formation of DNA)
The mainstream method is the so-called Southern blotting method, which detects by . Conventionally, radioactive isotopes (
High-sensitivity analysis has been carried out using RI (RI), but there are problems such as RI is expensive, dangerous to handle, and has a short half-life, making it difficult to obtain a stable probe. From this point of view, non-RI labeled probes have been actively developed. These include the biotin-avidin method, the hapten labeling method, the labezyme POD method, the chemprobe method, and the like.
これらは短鎖のオリゴヌクレオチドをプローブとした場
合の標識物の立体障害による２本鎖形成のμ■害、ある
いは高価な酵素等の使用などの欠点を有する。さらに、
従来の検出法によるためサンプルＤＮＡの電気泳動、固
定化などの煩雑な操作を必要とするのが現状である。These methods have drawbacks such as steric hindrance of the label when a short oligonucleotide is used as a probe, resulting in double-strand formation, and the use of expensive enzymes. moreover,
Currently, conventional detection methods require complicated operations such as electrophoresis and immobilization of sample DNA.
（問題点を解決するための手段）
本発明は、上記問題点を解決するもので、高価なＲＩや
酵素等をマーカーとして使用することなしに、安価で、
ＤＮＡの固定化等の煩雑な操作なしに、簡易、迅速に特
定の塩基配列を有するＤＮＡを検出することを目的とし
てなされたものである。(Means for Solving the Problems) The present invention solves the above problems, and can be done at low cost without using expensive RI, enzymes, etc. as markers.
This method was developed for the purpose of simply and quickly detecting DNA having a specific base sequence without complicated operations such as immobilization of DNA.
本発明は、蛍光標識された一本鎖ＤＮＡプローブを、分
析検体中のＤＮＡと接触させて２本鎖ＤＮＡを形成させ
、２本鎖形成前の蛍光偏光と２本鎖形成後の蛍光偏光と
の変位を測定して検体中のＤＮＡに、プローブに対応す
る塩基配列が存在することを検出する方法である。In the present invention, a fluorescently labeled single-stranded DNA probe is brought into contact with DNA in an analysis sample to form double-stranded DNA, and fluorescence polarization before double-strand formation and fluorescence polarization after double-strand formation are detected. This method detects the presence of a base sequence corresponding to a probe in DNA in a sample by measuring the displacement of the probe.
本発明では、蛍光標識された一本鎖ＤＮＡプローブが、
例えば、ＤＮＡの３′末端に、アミノ基を少なくとも１
つ有するデオキシヌクレオチド誘導体を結合させ、更に
インチオシアネート基を少なくとも１つ有する蛍光色素
を結合したものが使用される四
本発明で使用するプローブの製造において、アミノ基を
少なくとも１つ有するデオキシヌクレオチド誘導体とし
ては、脂肪族１級アミンを有するものが反応性にすぐれ
ている。例として８−（６−アミノヘキシル）−アミノ
アデノシン−５′−トリフオスフェートや５−（３−ア
ミノアリル）−ウリジン−５′−トリフオスフェートな
どがあげられるが、これに限定されるものではない。In the present invention, a fluorescently labeled single-stranded DNA probe is
For example, at least one amino group is added to the 3' end of the DNA.
In the production of the probe used in the present invention, a deoxynucleotide derivative having at least one amino group is used, and a fluorescent dye having at least one inthiocyanate group is used. Those having an aliphatic primary amine have excellent reactivity. Examples include, but are not limited to, 8-(6-aminohexyl)-aminoadenosine-5'-triphosphate and 5-(3-aminoallyl)-uridine-5'-triphosphate. do not have.
また１本発明において、イソチオシアネート基を少なく
とも１つ有する蛍光色素としては、フルオレッセインイ
ソチオシアネート（ＦＩＴＣ） 、テトラメチルローダ
ミンイソチオシアネート（ＴＲＩＴＣ）、エオシンイン
チオシアネート、などがあげられる。In the present invention, examples of the fluorescent dye having at least one isothiocyanate group include fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and eosin inthiocyanate.
ＤＮＡの３′末端にアミノ基を少なくとも１つ有するデ
オキシヌクレオチド誘導体を結合させる場合は、デオキ
シヌクレオチジルトランスフェラーゼ又はポリアデニレ
ートポリメラーゼを用いるのが好ましい。When bonding a deoxynucleotide derivative having at least one amino group to the 3' end of DNA, it is preferable to use deoxynucleotidyl transferase or polyadenylate polymerase.
ＤＮＡはアミノ基を少なくとも１つ有するデオキシヌク
レオチド誘導体及びデオキシヌクレオチジルトランスフ
ェラーゼ又はポリアデニレートポリメラーゼを反応させ
た後、酵素及び未反応のデオキシヌクレオチド誘導体を
分離除去する。After DNA is reacted with a deoxynucleotide derivative having at least one amino group and deoxynucleotidyl transferase or polyadenylate polymerase, the enzyme and unreacted deoxynucleotide derivative are separated and removed.
酵素反応は、リン酸緩衝液、カコジル酸緩衝液などを用
い、ＰＨ５〜９程度、温度３０〜４０℃程度で行なわれ
る。The enzyme reaction is carried out at a pH of about 5 to 9 and a temperature of about 30 to 40°C using a phosphate buffer, a cacodylate buffer, or the like.
酵素反応によって得られたアミノ基を少なくとも１つ有
するデオキシヌクレオチド誘導体を３′末端に結合した
ＤＮＡにはイソチオシアネート基を少なくとも１つ有す
る蛍光色素を加えて反応させ、未反応の蛍光色素を分離
除去することによって蛍光色素で標識されたＤＮＡを得
ることができる。A fluorescent dye having at least one isothiocyanate group is added to the DNA obtained by enzymatic reaction and has a deoxynucleotide derivative having at least one amino group attached to the 3' end, and the unreacted fluorescent dye is separated and removed. By doing so, DNA labeled with a fluorescent dye can be obtained.
本発明においては、プローブＤＮＡに標識された蛍光色
素の偏光を観測するこによりＤＮＡを検出することがで
きる。すなわち、蛍光色素を標識した短鎖のオリゴヌク
レオチドをプローブとして、検体中の特定の塩基配列を
持つＤＮＡを認識して２本鎖を形成し、これによって生
じる蛍光色素の偏光変化を測定することによって、特定
のＤＮＡを検出する。In the present invention, DNA can be detected by observing the polarization of the fluorescent dye labeled on the probe DNA. In other words, a short oligonucleotide labeled with a fluorescent dye is used as a probe to recognize DNA with a specific base sequence in the sample to form a double strand, and the resulting change in polarization of the fluorescent dye is measured. , detect specific DNA.
励起光を垂直方向に偏光してサンプルに照射した場合、
生じる蛍光は、励起光と同じ方向の吸収モーメントを持
つ分子のみが励起され、蛍光を発するためにその垂直成
分と水平成分の強度比が異って観測される、すなわち偏
光が生じる。しかしながら、通常この偏光は、励起分子
の急速な回転ブラウン運動によって緩和され、ａ測され
ないことが多い、短い一本鎖プローブＤＮＡに標識され
た蛍光色素を考えた場合、まずプローブＤＮＡが、大き
なサンプルＤＮＡ中の特定の塩基配列を認識して相補的
な２本鎖形成を行う。このみかけ上の分子量の増大によ
って標識された蛍光色素の速い回転運動が緩和されて、
偏光異方性が大きくなることが期待される。すなわち、
偏光度の変化を観測することによって、プローブＤＮＡ
とサンプルＤＮＡとの２本鎖形成、つまりプローブＤＮ
Ａと相補鎖を持つＤＮＡの検出が可能となる。もちろん
、サンプル中に相補鎖がない場合、プローブＤＮＡは２
本鎖を形成せず、したがって偏光の変化は観測されない
。When the excitation light is vertically polarized and irradiated onto the sample,
In the generated fluorescence, only molecules with an absorption moment in the same direction as the excitation light are excited, and because the fluorescence is emitted, the intensity ratio of the vertical and horizontal components is observed to be different, that is, polarized light is generated. However, this polarization is usually relaxed by the rapid rotational Brownian motion of the excited molecules and is often not measured.When considering a fluorescent dye labeled on a short single-stranded probe DNA, first the probe DNA is absorbed into a large sample. It recognizes a specific base sequence in DNA and forms a complementary double strand. This apparent increase in molecular weight relaxes the fast rotational motion of the labeled fluorescent dye,
It is expected that the polarization anisotropy will increase. That is,
By observing changes in the degree of polarization, the probe DNA
and sample DNA to form a double strand, that is, probe DNA
It becomes possible to detect DNA having a complementary strand to A. Of course, if there is no complementary strand in the sample, the probe DNA
No main chains are formed and therefore no change in polarization is observed.
（発明の効果）
本発明により、従来サザンブロッティング法など煩雑な
操作を必要としていた臨床分野におけるＤＮＡの診断を
迅速、簡易に行うことが可能となる。すなわちＤＮＡの
変異、あるいは感染症を誘発するウィルス等の外因性遺
伝子を迅速、簡易に検出することにより、各種の病気の
初期段階での診断、予防、治療が可能となる。(Effects of the Invention) The present invention makes it possible to quickly and easily perform DNA diagnosis in the clinical field, which conventionally required complicated operations such as Southern blotting. That is, by quickly and easily detecting DNA mutations or exogenous genes such as viruses that induce infectious diseases, it becomes possible to diagnose, prevent, and treat various diseases in their early stages.
（実施例）
実施例１
重合度１２−１８のオリゴアデノシンをプローブＤＮＡ
として、３′末端にターミナルデオキシヌクレオチジル
トランスフェラーゼを用いて８−（６−アミノヘキシル
）−アミノ−５′−トリフオスフェートを重合し、さら
にフルオレッセインイソチオシアネートを反応させて蛍
光色素を４Ｌａｔ、た。このプローブＤＮＡに相補鎖を
持つポリチミジンを添加し、４０℃、３０分間２本鎖形
成を行った。第１図にポリチミジンの添加量の増加に対
する蛍光異方性比を示した。プローブであるオリゴアデ
ノシンが大きなポリチミジンと２本鎖形成を行い標識さ
れた蛍光色素の急速な回転ブラウン運動が緩和されたた
めに偏光異方性比が増大した。従って、この偏光異方性
比の増大により、プローブの２本鎖形成が確認された。(Example) Example 1 Oligoadenosine with a degree of polymerization of 12-18 was used as a probe DNA
8-(6-aminohexyl)-amino-5'-triphosphate was polymerized at the 3' end using terminal deoxynucleotidyl transferase, and further reacted with fluorescein isothiocyanate to convert the fluorescent dye into 4Lat, Ta. Polythymidine having a complementary strand was added to this probe DNA, and double strand formation was performed at 40°C for 30 minutes. FIG. 1 shows the fluorescence anisotropy ratio with increasing amount of polythymidine added. The oligoadenosine probe formed a double strand with a large polythymidine, and the rapid rotational Brownian motion of the labeled fluorescent dye was relaxed, resulting in an increase in the polarization anisotropy ratio. Therefore, the formation of double strands of the probe was confirmed by this increase in polarization anisotropy ratio.
実施例２
プラスミドｐＢＲ３２２の検出を行った。プローブＤＮ
ＡとしてｐＢＲ３２２中の２０塩基（６２０−６３９）
の配列を持つオリゴヌクレオチドを
ＣＹＣＬＯＮＥ ＤＮＡ ５ＹＮＴＨＥＳＩＺＥＲ（Ｂ
ｉｏｓｅａｒｃｈ、 Ｉｎｃ、 ）で合成して使用した
。サンプルのｐＢＲ３２２は制限酵素ＥｃｏＲＩにより
７ｍＭ ＭｇＣｌ、と１２５ｍＭ ＮａＣ１を含む１０
ｍＭＴｒｉｓ−ＨＣＩ緩衝液（ｐＨ７，５）中で２４時
間反応させて直鎖のＤＮＡとした。また、このｐＢＲ３
２２とλ−ＤＮＡのそれぞれの水溶液を１５分間煮沸し
、急冷することによって−本領ＤＮＡサンプルとした。Example 2 Plasmid pBR322 was detected. Probe DN
20 bases (620-639) in pBR322 as A
CYCLONE DNA 5YNTHESIZER (B
It was synthesized and used by iosearch, Inc.). The sample pBR322 was purified with restriction enzyme EcoRI to contain 7mM MgCl and 125mM NaCl.
The DNA was reacted for 24 hours in mMTris-HCI buffer (pH 7,5) to obtain linear DNA. Also, this pBR3
The respective aqueous solutions of 22 and λ-DNA were boiled for 15 minutes and rapidly cooled to prepare the original DNA samples.
実施例１と同様に蛍光色素を標識した合成プローブＤＮ
Ａを各サンプルＤＮＡにそれぞれ加え、５ｘｓｓＰＥ緩
衝液（ｐＨ８，０）中で、６０℃、４時間ハイブリダイ
ゼーションを行った。表１に得られた偏光異方性を示す
、ブランクあるいはプローブＤＮＡと相補鎖を持たない
λ−ＤＮＡに比べてｐＢＲ３２２ではより大きな偏光異
方性が得られた。すなわちこの偏光異方性の増大により
ｐＢＲ３２２の検出ができた。Synthetic probe DN labeled with a fluorescent dye as in Example 1
A was added to each sample DNA, and hybridization was performed at 60°C for 4 hours in 5xssPE buffer (pH 8,0). A larger polarization anisotropy was obtained in pBR322 compared to the blank or the λ-DNA having no complementary strand to the probe DNA, which shows the polarization anisotropy obtained in Table 1. That is, pBR322 could be detected due to this increase in polarization anisotropy.
第１表
本発明のＤＮＡ迅速、簡易検出法は、プローブとして用
いたオリゴヌクレオチドに標識された蛍光色素の偏光異
方性を測定するものであり、この変化により、特定の塩
基配列を有するＤＮＡの検出が可能となった。Table 1 The rapid and simple DNA detection method of the present invention measures the polarization anisotropy of a fluorescent dye labeled on an oligonucleotide used as a probe, and this change allows the detection of DNA with a specific base sequence. Detection is now possible.
第１図は、本発明においてオリゴアデノシンをプローブ
とした場合の、ポリチミジンの添加量に対する偏光異方
性変化を示したものである。
代理人 弁理士 戸 １）親 男FIG. 1 shows the change in polarization anisotropy with respect to the amount of polythymidine added when oligoadenosine is used as a probe in the present invention. Agent Patent attorney 1) Parent Male
Claims (1)
中のＤＮＡと接触させて２本鎖ＤＮＡを形成させ、２本
鎖形成前の蛍光偏光と２本鎖形成後の蛍光偏光との変位
を測定して検体中のＤＮＡに、プローブに対応する塩基
配列が存在することを検出する方法。 ２、蛍光標識された一本鎖ＤＮＡプローブが、ＤＮＡの
３′末端に、アミノ基を少なくとも１つ有するデオキシ
ヌクレオチド誘導体を結合させ、更にイソチオシアネー
ト基を少なくとも１つ有する蛍光色素を結合させたもの
であることを特徴とする特許請求の範囲第１項記載の方
法。[Claims] 1. A fluorescently labeled single-stranded DNA probe is brought into contact with DNA in an analysis sample to form double-stranded DNA, and fluorescence polarization before double-stranded formation and after double-stranded formation are detected. A method of detecting the presence of a base sequence corresponding to a probe in DNA in a specimen by measuring the displacement of the probe with respect to fluorescence polarization. 2. A fluorescently labeled single-stranded DNA probe in which a deoxynucleotide derivative having at least one amino group is bound to the 3' end of the DNA, and a fluorescent dye having at least one isothiocyanate group is bound to the 3' end of the DNA. The method according to claim 1, characterized in that:
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22757788A JPH0275958A (en) | 1988-09-13 | 1988-09-13 | Detection of dna |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP22757788A JPH0275958A (en) | 1988-09-13 | 1988-09-13 | Detection of dna |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH0275958A true JPH0275958A (en) | 1990-03-15 |
Family
ID=16863096
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP22757788A Pending JPH0275958A (en) | 1988-09-13 | 1988-09-13 | Detection of dna |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0275958A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0838199A (en) * | 1994-04-18 | 1996-02-13 | Becton Dickinson & Co | Fluorescent polarization detecting method of nucleic acid amplification |
| DE4418691A1 (en) * | 1994-05-28 | 1996-02-22 | Boehringer Mannheim Gmbh | 3 '- (4'-) non-radioactively labeled nucleosides and nucleotides with amino carboxylic acid, peptide or carboxylic acid spacer |
| US5939256A (en) * | 1992-11-27 | 1999-08-17 | Canon Kabushiki Kaisha | Detection of nucleic acid hybrid variation which interacts with double helix or with second reagent through double helix by charge transfer and probe for hybridizing with target nucleic acid |
| WO1999060158A1 (en) * | 1998-05-19 | 1999-11-25 | Laboratory Of Molecular Biophotonics | Solid phase for detecting nucleic acid and method for detecting nucleic acid |
| US6124342A (en) * | 1993-12-27 | 2000-09-26 | Canon Kk | Pharmaceutical compositions containing pyrylium compounds, pyrylium salts and process for manufacturing a medicament containing the aforesaid compounds |
| US6297008B1 (en) | 1996-10-03 | 2001-10-02 | Canon Kabushiki Kaisha | Process for detecting target nucleic acid, process for quantifying the same, and pyrylium compound for chemiluminescence analysis |
| WO2014089645A1 (en) | 2012-12-11 | 2014-06-19 | Web Equipamentos Elétricos S.A. - Motores | Drainage system for electric machines, and electric machines comprising same |
-
1988
- 1988-09-13 JP JP22757788A patent/JPH0275958A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5939256A (en) * | 1992-11-27 | 1999-08-17 | Canon Kabushiki Kaisha | Detection of nucleic acid hybrid variation which interacts with double helix or with second reagent through double helix by charge transfer and probe for hybridizing with target nucleic acid |
| US6124342A (en) * | 1993-12-27 | 2000-09-26 | Canon Kk | Pharmaceutical compositions containing pyrylium compounds, pyrylium salts and process for manufacturing a medicament containing the aforesaid compounds |
| US6242477B1 (en) | 1993-12-27 | 2001-06-05 | Canon Kabushiki Kaisha | Pharmaceutical compositions containing pyrylium compounds, pyrylium salts and process for manufacturing a medicament containing the aforesaid compounds |
| JPH0838199A (en) * | 1994-04-18 | 1996-02-13 | Becton Dickinson & Co | Fluorescent polarization detecting method of nucleic acid amplification |
| DE4418691A1 (en) * | 1994-05-28 | 1996-02-22 | Boehringer Mannheim Gmbh | 3 '- (4'-) non-radioactively labeled nucleosides and nucleotides with amino carboxylic acid, peptide or carboxylic acid spacer |
| US6297008B1 (en) | 1996-10-03 | 2001-10-02 | Canon Kabushiki Kaisha | Process for detecting target nucleic acid, process for quantifying the same, and pyrylium compound for chemiluminescence analysis |
| WO1999060158A1 (en) * | 1998-05-19 | 1999-11-25 | Laboratory Of Molecular Biophotonics | Solid phase for detecting nucleic acid and method for detecting nucleic acid |
| WO2014089645A1 (en) | 2012-12-11 | 2014-06-19 | Web Equipamentos Elétricos S.A. - Motores | Drainage system for electric machines, and electric machines comprising same |
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