JP2002291500A - Method for detecting mutation of nucleic acid using nucleic acid melt curve and nucleic acid dissociation curve - Google Patents
Method for detecting mutation of nucleic acid using nucleic acid melt curve and nucleic acid dissociation curveInfo
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- JP2002291500A JP2002291500A JP2001135556A JP2001135556A JP2002291500A JP 2002291500 A JP2002291500 A JP 2002291500A JP 2001135556 A JP2001135556 A JP 2001135556A JP 2001135556 A JP2001135556 A JP 2001135556A JP 2002291500 A JP2002291500 A JP 2002291500A
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- nucleic acid
- mutation
- dna
- dissociation
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Abstract
Description
【0001】[0001]
【発明の属する技術分野】本発明は、2本鎖DNAを加
熱し、1本鎖に解離する温度の差異及びその特徴から2
本鎖DNAの変異を推定するDNA判別法に関する。TECHNICAL FIELD The present invention relates to a method for heating a double-stranded DNA and dissociating the double-stranded DNA into single strands based on the difference in temperature and the characteristics thereof.
The present invention relates to a DNA discrimination method for estimating a mutation of a single-stranded DNA.
【0002】[0002]
【従来の技術】2本鎖DNAの変異を検出する方法とし
ては、従来から様々な方法が考案され、実用化されてき
た。このDNAの変異とは、主にその塩基配列の塩基の
変異、または長さの変異に関するものであり、それらを
検出することにより判別されてきた。2. Description of the Related Art Various methods for detecting mutations in double-stranded DNA have been conventionally devised and put into practical use. The mutation in the DNA mainly relates to a mutation in the base of the base sequence or a mutation in the length thereof, and has been distinguished by detecting them.
【0003】塩基配列の変異の検出法としては、塩基の
変異のある部分に意図的にPCRプライマーの3’末端
を配置し、PCRが行われるか否かで判別するPCR−
ASP(Allele Specific Prime
r)法や、変異部分をPCRで増幅し、これが制限酵素
で切断可能か否かで判別するPCR−RFLP(Res
triction Fragment Length
Polymorphism)法がある。[0003] As a method for detecting a mutation in a base sequence, the 3 'end of a PCR primer is intentionally placed at a portion where a base mutation is present, and a PCR-based method is used to determine whether or not PCR is performed.
ASP (Allele Specific Prime)
r) method or a PCR-RFLP (Res) in which a mutated portion is amplified by PCR and discriminated based on whether it can be cleaved with a restriction enzyme.
trickion Fragment Length
(Polymorphism) method.
【0004】この他、PCR産物を意図的に変性せず電
気泳動を行い、変異で生じる立体構造の差に起因する泳
動度の変化を検出するPCR−SSCP(Single
Strand Conformation Poly
morphism)法、変異部分をさらに蛍光標識プロ
ーブで検出するタックマンプローブ法なども汎用されて
いる。[0004] In addition, PCR-SSCP (Single), which performs electrophoresis without intentionally denaturing a PCR product and detects a change in electrophoretic mobility caused by a difference in three-dimensional structure caused by the mutation.
Strand Conformation Poly
Morphism) method, and a Taqman probe method in which a mutated portion is further detected with a fluorescent-labeled probe are also widely used.
【0005】最近では上述の方法に加え、全ての塩基配
列を直接確認するシークエンス法、DNAチップ法など
の方法を用いて遺伝子の塩基配列の変異を検出する方法
も行われている。In recent years, in addition to the above-mentioned methods, a method of directly detecting all nucleotide sequences, a method of detecting a mutation in a nucleotide sequence of a gene by using a method such as a DNA chip method and the like has also been carried out.
【0006】塩基配列の長さの変異はマイクロサテライ
トの検出法として、同じく様々な方法が行われている。
PCR−RFLP法、PCR−SSCP法、シークエン
ス法などはその代表的な方法として知られている。[0006] Variations in the length of the base sequence have been similarly performed by various methods for detecting microsatellite.
The PCR-RFLP method, the PCR-SSCP method, the sequencing method and the like are known as typical methods.
【0007】[0007]
【発明が解決しようとする課題】現在までに行われてい
る核酸変異検出法は、いずれも熟練した技術を必要と
し、かつ検出には相応の時間とコストを必要とする。唯
一、タックマンプローブ法はリアルタイム定量PCR法
を応用した技術であるため、迅速な核酸変異の検出が可
能であるが、蛍光標識プローブが高価であること、未知
の変異に対応出来ないことやプローブの設計に高度の技
術を要するなどの難点がある。All of the nucleic acid mutation detection methods that have been carried out so far require skilled techniques, and the detection requires a considerable amount of time and cost. The only method that uses the real-time quantitative PCR method is the TaqMan probe method, which enables rapid detection of nucleic acid mutations.However, fluorescent labeled probes are expensive, cannot cope with unknown mutations, There are drawbacks such as the need for advanced technology for design.
【0008】何れの方法も上記に記したように、技術・
コスト・時間の全てを満足させる検出法ではなく、つま
り、高度な技術を必要とせず、短時間に安価に核酸の変
異を検出する方法が開発されていないのが現状である。[0008] As described above, each of the methods is a technology and
At present, it is not a detection method that satisfies all of cost and time, that is, a method for detecting a mutation of a nucleic acid in a short time and at low cost without requiring advanced technology has been developed.
【0009】[0009]
【課題を解決するための手段】係る事情に鑑み発明者は
鋭意研究の結果、2本鎖DNA、又はその増幅産物を加
熱し、1本鎖に解離する温度点の差異及びその特徴から
2本鎖DNAの変異を短時間に容易に推定する核酸変異
検出法の発明に至った。Means for Solving the Problems In view of the above circumstances, the present inventors have conducted intensive studies and as a result, have found that the difference between the temperature points at which double-stranded DNA or its amplification product is heated and dissociated into single-stranded DNA, and the characteristics thereof, The invention of a nucleic acid mutation detection method for easily estimating the mutation of a strand DNA in a short time has been attained.
【0010】本発明の原理を説明すれば、検体中の2本
鎖DNAのPCR産物に温度をかけていくと、それらは
各々1本鎖DNAに解離する。この急激に解離する温度
点をTm(Melting Temperature)
値と呼ぶが、解離温度及びスピードなどの特性は2本鎖
DNAの塩基配列及び塩基のアデニン(A)とチミン
(T)間、グアニン(G)とシトシン(C)間の結合強
度に差があるために各塩基A、G、C、Tの含有率に左
右される。又、この解離する温度及びスピードは同じく
PCR産物の長さにも左右されるため、マイクロサテラ
イトのリピート数の差異によっても変化する。従って予
め野生型(正常型)のTm値あるいはTmのパターンを
作成しておけば、検体DNAのTm値あるいはTmのパ
ターンを比較することにより、変異型(異常型)の存在
を検出することが可能である。To explain the principle of the present invention, when a temperature is applied to a double-stranded DNA PCR product in a specimen, each of them dissociates into single-stranded DNA. This rapidly dissociating temperature point is defined as Tm (Melting Temperature).
The properties such as dissociation temperature and speed are different in the base sequence of double-stranded DNA and the binding strength between bases adenine (A) and thymine (T) and between guanine (G) and cytosine (C). This depends on the content of each base A, G, C, T. In addition, the temperature and speed of dissociation also depend on the length of the PCR product, and thus vary depending on the difference in the number of microsatellite repeats. Therefore, if the Tm value or Tm pattern of the wild type (normal type) is prepared in advance, the presence of the mutant type (abnormal type) can be detected by comparing the Tm value or Tm pattern of the sample DNA. It is possible.
【0011】前記の発明原理を図面を用いて説明すると
図1及び図2の様になる。図1は横軸に温度、縦軸に蛍
光強度をとって示した核酸溶解曲線であるが、例えば
A、B、Cの三種のPCR産物に温度をかけていくと2
本鎖DNAは一定の温度で1本鎖DNAに解離していく
ために蛍光強度が徐々に低下していくが、この低下のパ
ターンはPCR産物中のGC含有量、長さ、立体構造な
どにより規定されて各々独自の曲線となる。ここで急激
に解離の進行する温度を解離温度(Tm値)と呼ぶ。The principle of the invention will be described with reference to the drawings as shown in FIGS. FIG. 1 is a nucleic acid dissolution curve in which temperature is plotted on the horizontal axis and fluorescence intensity is plotted on the vertical axis. For example, when temperature is applied to three types of PCR products A, B and C, 2
The fluorescence intensity gradually decreases due to the dissociation of single-stranded DNA into single-stranded DNA at a certain temperature, but the pattern of this decrease depends on the GC content, length, three-dimensional structure, etc. in the PCR product. Defined and each has its own curve. Here, the temperature at which the dissociation proceeds rapidly is called the dissociation temperature (Tm value).
【0012】図1を微分したものが図2に示す核酸解離
曲線である。この図ではTm値はピークとして表示され
るので、曲線間の違いが一層明らかとなる。PCR産物
は各々独自の解離曲線を持つので予め実験的に作成して
おいた正常な核酸の解離曲線と比較することによって核
酸変異を検出するのが本発明の原理となっている。The nucleic acid dissociation curve shown in FIG. 2 is obtained by differentiating FIG. In this figure, the Tm value is displayed as a peak, so that the difference between the curves becomes more apparent. Since each PCR product has its own dissociation curve, the principle of the present invention is to detect a nucleic acid mutation by comparing it with a dissociation curve of a normal nucleic acid which has been experimentally prepared in advance.
【0013】本発明は、採取された検体からゲノムDN
Aを抽出してDNA溶解曲線あるいはDNA解離曲線、
又は核酸増幅を実施することによって核酸増幅物の溶解
曲線あるいは解離曲線を作成してTm値を求める方法以
外に検体を破壊する構成物、酵素反応阻害因子を抑制す
る構成物などを含むダイレクトPCRバッファーなどを
使用することによって検体からDNAを抽出することな
く、直接PCRを行って核酸増幅を行う方法においても
本発明は有効である。[0013] The present invention provides a method for preparing a genomic DN from a collected specimen.
A is extracted and a DNA dissolution curve or a DNA dissociation curve,
Or a direct PCR buffer containing a component that destroys a sample, a component that suppresses an enzyme reaction inhibitor, and the like in addition to a method for obtaining a Tm value by preparing a dissolution curve or a dissociation curve of a nucleic acid amplification product by performing nucleic acid amplification The present invention is also effective in a method of performing nucleic acid amplification by directly performing PCR without extracting DNA from a sample by using such a method.
【0014】検体からDNAを抽出することなく、直接
PCRで核酸増幅を行う場合のダイレクトPCRバッフ
ァーとして株式会社島津製作所のAmpdirectが
有用であるが、本発明はこれに限定されるものではな
い。[0014] Ampdirect from Shimadzu Corporation is useful as a direct PCR buffer when nucleic acid amplification is performed by direct PCR without extracting DNA from the sample, but the present invention is not limited to this.
【0015】[0015]
【発明の実施の形態】次に、本発明の核酸変異検出法
を、既知の核酸を試料に用いて表1に示したPCR反応
液の処方で詳細に説明する。BEST MODE FOR CARRYING OUT THE INVENTION Next, the nucleic acid mutation detection method of the present invention will be described in detail by using a known nucleic acid as a sample and formulating a PCR reaction solution shown in Table 1.
【0016】[0016]
【表1】 [Table 1]
【0017】試料の検体DNAを入れて合計が50μl
となるように処方した表1に示した反応液を良く攪拌
し、その全量50μlをABI社製のリアルタイムPC
R定量機GeneAmp5700にセットしてPCRを
開始する。PCRプロトコールは、98℃/1秒、55
℃/10秒、72℃/10秒を40サイクル行う。A total of 50 μl including the sample DNA of the sample
The reaction solution shown in Table 1 was thoroughly stirred, and a total volume of 50 μl was transferred to a real-time PC manufactured by ABI.
The PCR is started by setting the R quantifier GeneAmp5700. The PCR protocol was 98 ° C / 1 second, 55
C./10 seconds and 72.degree. C./10 seconds are performed for 40 cycles.
【0018】PCR終了後、DNA溶解曲線(Melt
Curve)、および該曲線を微分して得られたDN
A解離曲線(Dissociation Curve)
が作成されるが、これらを予め実験的に作成した野生型
のDNA溶解曲線およびDNA解離曲線と比較して検体
DNAの変異の有無を検出する。尚、リアルタイムPC
R定量機は限定されるものでなく何れの製品でも構わな
い。又、PCRプロトコールは使用プライマーに合わせ
て適宜変更して実施する。After completion of the PCR, the DNA dissolution curve (Melt
Curve) and DN obtained by differentiating the curve.
A dissociation curve (Dissociation Curve)
These are compared with a wild-type DNA dissolution curve and a DNA dissociation curve, which have been experimentally prepared in advance, to detect the presence or absence of a mutation in the sample DNA. In addition, real-time PC
The R quantifier is not limited, and any product may be used. In addition, the PCR protocol is appropriately changed and performed according to the primers used.
【0019】[0019]
【実施例1】ヒトGlyceraldehyde−3−
Phosphate Dehydrogenase(G
3PDH)を野生型、マウスG3PDHを変異型と仮定
し、両者のTm値およびTmのパターンを比較した。Example 1 Human Glyceraldehyde-3-
Phosphate Dehydrogenase (G
Assuming that 3PDH) was a wild type and mouse G3PDH was a mutant type, the Tm values and Tm patterns of both were compared.
【0020】ヒトおよびマウスのゲノムDNAを、抽出
試薬として日本ジーン社のIsogen−LSを使用し
て常法に従い抽出後、表1の処方に従い反応液を調整
し、各々のゲノムDNA1μgおよび下記塩基配列のプ
ライマー、 プライマー1:5’−ACCACAGTCCATGCC
ATCAC−3’(塩基配列1)、 プライマー2:5’−TCCACCACCCTGTTG
CTGTA−3’(塩基配列2)、 を使用してABI社製のリアルタイムPCR定量機Ge
neAを使用して98℃/1秒、55℃/10秒、72
℃/10秒を40サイクルのプロトコールでPCRを行
った。検討は各サンプルにつき夫々6回行い、得られた
結果を表2に示す。Human and mouse genomic DNAs were extracted according to a conventional method using Isogen-LS of Nippon Gene Co., Ltd. as an extraction reagent, and the reaction solutions were prepared according to the prescription shown in Table 1. Primer 1: Primer 1: 5'-ACCACAGTCCATGCCC
ATCAC-3 '(base sequence 1), Primer 2: 5'-TCCACCACCCTGTTG
CTGTA-3 '(base sequence 2), using a real-time PCR quantifier Ge manufactured by ABI
98 ° C./1 second, 55 ° C./10 seconds, 72
PCR was performed according to a protocol of 40 cycles at / 10 ° C. The examination was performed six times for each sample, and the obtained results are shown in Table 2.
【0021】[0021]
【表2】 [Table 2]
【0022】表2から正常であるヒト(野生型)のTm
値に一つのピークが90.5℃付近に観測されるが、マ
ウス(変異型)のTm値は二つのピークが観測され、そ
のピーク温度もヒト(野性型)のそれとは明らかに異な
っていることからこの核酸は変異をしていることが判
る。Table 2 shows that the normal human (wild-type) Tm
One peak is observed at around 90.5 ° C., but two peaks are observed in the Tm value of the mouse (mutant type), and the peak temperatures are clearly different from those of the human (wild type). This indicates that this nucleic acid is mutated.
【0023】[0023]
【実施例2】ヒトG3PDHを野生型、これに対し過量
の塩化マグネシウム(12μl:final 6mM)
を反応液に添加したものを非特異的核酸増幅モデルとし
両者のTm値およびTmのパターンを比較した。但し、
非特異的核酸増幅モデルでは増量した塩化マグネシウム
量に対して総量を水で調整した。[Example 2] Human G3PDH was used as a wild type, whereas an excessive amount of magnesium chloride (12 µl: final 6 mM) was used.
Was used as a non-specific nucleic acid amplification model, and the Tm values and Tm patterns of both were compared. However,
In the non-specific nucleic acid amplification model, the total amount was adjusted with water with respect to the increased amount of magnesium chloride.
【0024】ヒトゲノムDNAを、抽出試薬として日本
ジーン社のIsogen−LSを使用して常法に従い抽
出後、表1の処方に従い反応液を調整し、各々のゲノム
DNA1μgおよび前記塩基配列1及び2のプライマー
を使用してABI社製のリアルタイムPCR定量機Ge
neAを使用して98℃/1秒、55℃/10秒、72
℃/10秒を40サイクルのプロトコールでPCRを行
った。検討は各サンプルにつき夫々6回行い、得られた
結果を表3に示す。After extracting human genomic DNA using Isogen-LS of Nippon Gene as an extraction reagent according to a conventional method, a reaction solution was prepared according to the prescription shown in Table 1, and 1 μg of each genomic DNA and the above-mentioned nucleotide sequences 1 and 2 were prepared. ABI real-time PCR quantifier Ge using primers
98 ° C./1 second, 55 ° C./10 seconds, 72
PCR was performed according to a protocol of 40 cycles at / 10 ° C. The examination was performed six times for each sample, and the obtained results are shown in Table 3.
【0025】[0025]
【表3】 [Table 3]
【0026】表3の結果から塩化マグネシウムをPCR
反応液に過量添加して非特異的核酸増幅したモデルでは
正常であるヒト(野生型)のTm値と明らかに異なった
核酸解離曲線を示しており、変異を容易に検出出来る。From the results in Table 3, magnesium chloride was subjected to PCR.
In a model in which non-specific nucleic acid amplification was performed by adding an excess amount to the reaction solution, a nucleic acid dissociation curve clearly different from that of a normal human (wild type) Tm value was shown, and mutations could be easily detected.
【0027】[0027]
【実施例3】ヒトG3PDHを野生型、これに対し過量
のプライマー(各10μl:final 100pmo
l)を反応液に添加したものをプライマー二量体形成モ
デルとし両者のTm値およびTmのパターンを比較し
た。実施例2と同様の方法でリアルタイムPCRを行っ
た結果を表4に示す。Example 3 Human G3PDH was used as a wild type, and an excess amount of primer (10 μl each: final 100 pmo)
l) was added to the reaction solution, and the resulting mixture was used as a primer dimer formation model to compare the Tm values and Tm patterns of the two. Table 4 shows the results of performing real-time PCR in the same manner as in Example 2.
【0028】[0028]
【表4】 [Table 4]
【0029】表4からPCR反応液にプライマーを過量
に添加してプライマー二量体を形成した場合には正常の
場合のTm値と異なる温度にピークが観測される。この
差はDNA中のA=TとG=Cの結合力の差に起因する
もので、プライマー二量体形成モデルの方がG=C含有
量が少ないために低い温度で解離する。このことから変
異を容易に検出することが出来る。From Table 4, when the primer is excessively added to the PCR reaction solution to form a primer dimer, a peak is observed at a temperature different from the Tm value in a normal case. This difference is due to the difference in the binding force between A = T and G = C in DNA, and the primer dimer formation model dissociates at a lower temperature due to the lower G = C content. From this, the mutation can be easily detected.
【0030】[0030]
【発明の効果】本法は、検体DNAのTm値あるいはT
mのパターンを比較するだけで、既知の核酸変異が塩基
置換変異、塩基数変異とも容易に短時間で、しかも安価
に検出が可能となる。更に、未知の核酸変異についても
パターンを群別化することにより推定が可能となる。こ
の他、外来遺伝子の混入があった場合、プライマーに二
量体が形成された場合、非特異的な核酸増幅が行われた
場合など、核酸増幅過程に何らかの問題があった場合も
Tm値あるいはTmのパターンに変化が見られるため、
核酸増幅が正常に行われたか否かの判定も可能となる。
本発明を従来の方法と比較すると表5の様になる。According to the present method, the Tm value or T
By simply comparing the patterns of m, it is possible to easily detect both known nucleic acid mutations, base substitution mutations and base number mutations in a short time and at low cost. Furthermore, unknown nucleic acid mutations can be estimated by grouping patterns. In addition, when there is any problem in the nucleic acid amplification process, such as when a foreign gene is mixed, when a dimer is formed on the primer, when nonspecific nucleic acid amplification is performed, the Tm value or Because a change is seen in the pattern of Tm,
It is also possible to determine whether nucleic acid amplification has been performed normally.
Table 5 shows a comparison of the present invention with the conventional method.
【0031】[0031]
【表5】 [Table 5]
【0032】表5の通りPCR−ASP法などの従来方
法に比較して本発明の方法は検出時間や検出コストに優
れ、しかも特別な技術を習得する必要でない技術である
ことが明らかである。As shown in Table 5, it is clear that the method of the present invention is superior to the conventional method such as the PCR-ASP method in the detection time and the detection cost, and does not require special techniques.
【0033】[0033]
【配列表】SEQUENCE LISTING 〈110〉大島 譲二 〈120〉核酸溶解曲線及び核酸解離曲線を用いた核酸
変異検出法 〈130〉OJ010329 〈141〉2001−3−29 〈160〉2 〈210〉1 〈211〉20 〈212〉DNA 〈213〉Artificial Sequence 〈400〉1 accacagtcc atgccatcac 20 〈210〉2 〈211〉DNA 〈213〉Artificial Sequence 〈400〉2 tccaccaccc tgttgctgta 20[Sequence list] SEQUENCE LISTING <110> Joji Oshima <120> Nucleic acid mutation detection method using nucleic acid dissolution curve and nucleic acid dissociation curve <130> OJ010329 <141> 2001-3-29 <160> 2 <210> 1 <211 > 20 <212> DNA <213> Artificial Sequence <400> 1 accacagtcc atgccatcac 20 <210> 2 <211> DNA <213> Artificial Sequence <400> 2
【図1】温度に対する蛍光強度の関係で示した核酸溶解
曲線の一例である。FIG. 1 is an example of a nucleic acid dissolution curve represented by a relationship between a fluorescence intensity and a temperature.
【図2】核酸溶解曲線を微分して得られた核酸解離曲線
の一例である。FIG. 2 is an example of a nucleic acid dissociation curve obtained by differentiating a nucleic acid dissolution curve.
Claims (12)
ノムDNAを抽出後リアルタイム定量PCR法で増幅
し、野生型ゲノムDNAを用いて予め実験的に作成して
おいたPCR産物溶解曲線(Melt Curve)、
あるいは該曲線を微分して得られたPCR産物解離曲線
(Dissociation Curve)との比較か
らゲノムDNAの変異を推定することを特徴とする核酸
変異検出法。1. A method for detecting mutation of a nucleic acid, comprising extracting genomic DNA from a sample, amplifying the extracted genomic DNA by real-time quantitative PCR, and experimentally preparing a PCR product dissolution curve using wild-type genomic DNA. Melt Curve),
Alternatively, a nucleic acid mutation detection method characterized by estimating a mutation in genomic DNA from a comparison with a PCR product dissociation curve (Dissociation Curve) obtained by differentiating the curve.
ノムDNAを核酸増幅法で増幅し、野生型ゲノムDNA
を用いて予め実験的に作成しておいた核酸増幅産物溶解
曲線(Melt Curve)、あるいは該曲線を微分
して得られる核酸増幅産物解離曲線(Dissocia
tion Curve)との比較からゲノムDNAの変
異を推定することを特徴とする核酸変異検出法。2. A method for detecting mutation of a nucleic acid, comprising the steps of: amplifying genomic DNA from a sample by a nucleic acid amplification method;
Nucleic acid amplification product dissolution curve (Melt Curve) that has been experimentally prepared in advance using, or a nucleic acid amplification product dissociation curve (Dissocia) obtained by differentiating the curve.
A method for detecting a nucleic acid mutation, comprising estimating a genomic DNA mutation from a comparison with the T.C.
ノムDNAを直接抽出し、野生型ゲノムDNAを用いて
あらかじめ実験的に作成しておいたDNA溶解曲線(M
elt Curve)、あるいは該曲線を微分して得ら
れたDNA解離曲線(Dissociation Cu
rve)との比較からゲノムDNAの変異を推定するこ
とを特徴とする核酸変異検出法。3. A method for detecting a mutation of a nucleic acid, wherein genomic DNA is directly extracted from a sample, and a DNA melting curve (M
elt Curve) or a DNA dissociation curve (Dissociation Cu) obtained by differentiating the curve.
a method for detecting a mutation in a nucleic acid, comprising estimating a mutation in genomic DNA from comparison with rve).
となく、ダイレクトPCRバッファーなどを併用し、直
接リアルタイム定量PCR法で増幅し、野生型ゲノムD
NAを用いて予め実験的に作成しておいたPCR産物溶
解曲線(Melt Curve)、あるいは該曲線を微
分して得られたPCR産物解離曲線(Dissocia
tion Curve)との比較からゲノムDNAの変
異を推定することを特徴とする核酸変異検出法。4. A method for directly amplifying wild-type genome D by real-time quantitative PCR using a direct PCR buffer or the like without extracting genomic DNA from the sample.
A PCR product dissolution curve (Melt Curve) experimentally prepared in advance using NA, or a PCR product dissociation curve (Dissocia) obtained by differentiating the curve.
A method for detecting a nucleic acid mutation, comprising estimating a genomic DNA mutation from a comparison with the T.C.
となく、ダイレクトバッファーなどを併用し、核酸増幅
法で増幅し、野生型ゲノムDNAを用いて予め実験的に
作成しておいた核酸増幅産物溶解曲線(Melt Cu
rve)、あるいは該曲線を微分して得られた核酸増幅
産物解離曲線(Dissociation Curv
e)との比較からゲノムDNAの変異を推定することを
特徴とする核酸変異検出法。5. A method for dissolving a nucleic acid amplification product which has been amplified by a nucleic acid amplification method using a direct buffer or the like without extracting genomic DNA from the sample, and which has been experimentally prepared in advance using wild-type genomic DNA. Curve (Melt Cu
rve) or a nucleic acid amplification product dissociation curve (Dissociation Curv) obtained by differentiating the curve.
A method for detecting a nucleic acid mutation, comprising estimating a mutation in genomic DNA from the comparison with e).
となく、ダイレクトバッファーなどを併用し、野生型ゲ
ノムDNAを用いて予め実験的に作成しておいたDNA
溶解曲線(Melt Curve)、あるいは該曲線を
微分して得られたDNA解離曲線(Dissociat
ion Curve)との比較からゲノムDNAの変異
を推定することを特徴とする核酸変異検出法。6. A DNA which has been experimentally prepared in advance using wild-type genomic DNA in combination with a direct buffer or the like without extracting genomic DNA from the sample.
Melt curve or DNA dissociation curve (Dissociat) obtained by differentiating the curve.
(ion Curve), and a method for detecting a mutation in a nucleic acid, comprising estimating a mutation in genomic DNA based on comparison with ionic curve.
の方法を用いて、マイクロサテライトなど、長さの異な
る変異型の検出をDNA溶解曲線(MeltCurv
e)、あるいは該曲線を微分して得られたDNA解離曲
線(Dissociation Curve)との比較
からDNAの変異を推定することを特徴とする核酸変異
検出法。7. The method according to any one of claims 1 to 6, wherein detection of mutants having different lengths, such as microsatellite, is performed using a DNA melting curve (MeltCurv).
e) or a nucleic acid mutation detection method comprising estimating a DNA mutation from a comparison with a DNA dissociation curve (Dissociation Curve) obtained by differentiating the curve.
の方法を用いて、塩基配列の異なる変異型の検出をDN
A溶解曲線(Melt Curve)、あるいは該曲線
を微分して得られたDNA解離曲線(Dissocia
tion Curve)との比較からDNAの変異を推
定することを特徴とする核酸変異検出法。8. The method according to any one of claims 1 to 6, wherein the detection of a mutant having a different base sequence is carried out by DN.
A dissolution curve (Melt Curve) or a DNA dissociation curve (Dissociia) obtained by differentiating the curve.
A method for detecting a nucleic acid mutation, comprising estimating a DNA mutation based on comparison with the T. curve.
の方法を用いて、人為的に合成したDNAの変異の検出
をDNA溶解曲線(Melt Curve)、あるいは
該曲線を微分して得られたDNA解離曲線(Disso
ciation Curve)との比較から推定するこ
とを特徴とする核酸変異検出法。9. The method according to any one of claims 1 to 8, wherein the detection of the mutation of the artificially synthesized DNA is determined by differentiating the DNA melting curve (Melt Curve) or the curve. The obtained DNA dissociation curve (Disso
(Circulation Curve).
載の方法を用いて、RNAから逆転写法(RT−PCR
法など)で人為的に合成したDNAの変異の検出をDN
A溶解曲線(Melt Curve)、あるいは該曲線
を微分して得られたDNA解離曲線(Dissocia
tion Curve)との比較から推定することを特
徴とする核酸変異検出法。10. A reverse transcription method (RT-PCR) from RNA using the method according to any one of claims 1 to 8 above.
Detection of DNA mutations artificially synthesized by the
A dissolution curve (Melt Curve) or a DNA dissociation curve (Dissociia) obtained by differentiating the curve.
a method for detecting a nucleic acid mutation, which is inferred from a comparison with T.C.
載の方法を用いて、混入DNAなど外在性因子に起因す
る非特異的な反応の結果生じた予期せぬ核酸増幅産物の
検出をDNA溶解曲線(Melt Curve)、ある
いは該曲線を微分して得られたDNA解離曲線(Dis
sociation Curve)との比較から推定す
ることを特徴とする核酸変異検出法。11. The use of the method according to any one of claims 1 to 8, wherein an unexpected nucleic acid amplification product resulting from a nonspecific reaction caused by an exogenous factor such as contaminating DNA is obtained. Detection was performed using a DNA melting curve (Melt Curve) or a DNA dissociation curve (Diss) obtained by differentiating the curve.
and a nucleic acid mutation detection method characterized by estimating from a comparison with S.C.
載の方法を用いて、プライマー2量体形成、鋳型DNA
非特異的反応など、内在性因子に起因する非特異的な反
応の結果により生じた予期せぬ核酸増幅産物の検出を、
DNA溶解曲線(Melt Curve)、あるいは該
曲線を微分して得られたDNA解離曲線(Dissoc
iation Curve)との比較から推定すること
を特徴とする核酸変異検出法。12. A method for forming a primer dimer and a template DNA using the method according to any one of claims 1 to 8.
Detection of unexpected nucleic acid amplification products resulting from non-specific reactions caused by endogenous factors, such as non-specific reactions,
DNA melting curve (Melt Curve) or a DNA dissociation curve (Dissoc) obtained by differentiating the curve
a method for detecting a nucleic acid mutation, which is inferred from a comparison with a mutation curve (i.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7547514B2 (en) | 2004-07-28 | 2009-06-16 | Canon U.S. Life Sciences, Inc. | Methods for monitoring genomic DNA of organisms |
US7604938B2 (en) | 2005-02-18 | 2009-10-20 | Canon U.S. Life Sciences, Inc. | Devices and methods for monitoring genomic DNA of organisms |
-
2001
- 2001-03-29 JP JP2001135556A patent/JP2002291500A/en active Pending
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7547514B2 (en) | 2004-07-28 | 2009-06-16 | Canon U.S. Life Sciences, Inc. | Methods for monitoring genomic DNA of organisms |
US7604938B2 (en) | 2005-02-18 | 2009-10-20 | Canon U.S. Life Sciences, Inc. | Devices and methods for monitoring genomic DNA of organisms |
US8841093B2 (en) | 2005-02-18 | 2014-09-23 | Canon U.S. Life Sciences, Inc. | Devices and methods for monitoring genomic DNA of organisms |
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