JP4454265B2 - Melting curve analysis method - Google Patents
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Description
本発明は、核酸の融解曲線解析法に関する。 The present invention relates to a method for analyzing melting curves of nucleic acids.
融解曲線解析は、核酸プローブをターゲット核酸にハイブリダイズさせた後、徐々に温度を上昇させ、この昇温中に、ハイブリッドの融解により変化するシグナルを測定し、シグナルの変化により、核酸プローブとターゲット核酸とのハイブリッドの融解曲線を解析する方法として知られている。融解曲線の解析により得られる融解温度は、核酸プローブとターゲット核酸との相同性を反映するため、この融解温度の決定により、ターゲット核酸の同一性が判別できる。また、核酸プローブを一塩基多型の部分に設定した場合、融解温度の決定により多型の型を判別できる（例えば、非特許文献１参照）。
本発明の課題は、感度の高い融解曲線解析法およびその方法を実施するためのプログラムを提供することである。 An object of the present invention is to provide a melting curve analysis method with high sensitivity and a program for carrying out the method.
本発明者らは、融解曲線の測定を繰り返すことにより、ハイブリッドの融解によるシグナルの変化量が大きくなることを見出し、この知見に基づき本発明を完成した。 The present inventors have found that the amount of change in signal due to melting of the hybrid increases by repeating the measurement of the melting curve, and the present invention has been completed based on this finding.
本発明は、核酸プローブとターゲット核酸とのハイブリッドを含む溶液の温度を上昇させ、この昇温中に、ハイブリッドの融解により変化するシグナルを測定し、シグナルの変化により核酸プローブとターゲット核酸とのハイブリッドの融解曲線を解析することを含む融解曲線解析法であって、
ハイブリッドのシグナルの測定の前に、溶液の温度を、核酸プローブとターゲット核酸とのハイブリッドが形成される温度から、核酸プローブとターゲット核酸が解離する温度まで連続的に上昇させ、次いで、核酸プローブとターゲット核酸とのハイブリッドが形成する温度まで下降させる工程を行うことを特徴とする解析法を提供する。
The present invention increases the temperature of a solution containing a hybrid of a nucleic acid probe and a target nucleic acid, measures a signal that changes due to melting of the hybrid during this temperature increase, and hybridizes the nucleic acid probe and the target nucleic acid by changing the signal. A melting curve analysis method comprising analyzing a melting curve of
Prior to the measurement of the hybrid signal, the temperature of the solution is continuously increased from the temperature at which the nucleic acid probe and target nucleic acid are hybridized to the temperature at which the nucleic acid probe and target nucleic acid dissociate, providing an analysis method characterized by a step of lowering to a temperature of hybrids formed between the target nucleic acid.
本発明の方法においては、シグナルは蛍光であることが好ましい。 In the method of the present invention, the signal is preferably fluorescence.
また、本発明は、核酸プローブとターゲット核酸とのハイブリッドを含む溶液の温度を上昇させ、この昇温中に、ハイブリッドの融解により変化するシグナルを測定し、シグナルの変化により核酸プローブとターゲット核酸とのハイブリッドの融解曲線を解析することを含む融解曲線解析法を行うための、温度の制御が可能な恒温装置の温度を制御する温度制御手段としてコンピューターを機能させるためのプログラムであって、
温度制御手段は、ハイブリッドのシグナルの測定のための温度の制御の前に、溶液の温度を、核酸プローブとターゲット核酸とのハイブリッドが形成される温度から、核酸プローブとターゲット核酸が解離する温度まで連続的に上昇させ、次いで、核酸プローブとターゲット核酸とのハイブリッドが形成するまで下降させる温度の制御を行う、プログラムを提供する。
Further, the present invention increases the temperature of a solution containing a hybrid of a nucleic acid probe and a target nucleic acid, measures a signal that changes due to melting of the hybrid during this temperature increase, A program for causing a computer to function as a temperature control means for controlling the temperature of a thermostat capable of controlling temperature for performing a melting curve analysis method including analyzing a melting curve of the hybrid of
The temperature control means adjusts the temperature of the solution from the temperature at which the hybrid of the nucleic acid probe and the target nucleic acid is formed to the temperature at which the nucleic acid probe and the target nucleic acid are dissociated before controlling the temperature for measurement of the hybrid signal. A program is provided that controls the temperature that is continuously raised and then lowered until a hybrid of a nucleic acid probe and a target nucleic acid is formed.
本発明によれば、高感度な融解曲線解析(Tm解析)が可能になる。特に、一回のTm解析ではシグナルの変化量が十分でなく、融解温度の決定ができなかったり、その精度が低かったりするような場合であっても、正確なTm解析が可能になる。 According to the present invention, a highly sensitive melting curve analysis (Tm analysis) becomes possible. In particular, even if the amount of change in signal is not sufficient in a single Tm analysis and the melting temperature cannot be determined or the accuracy is low, accurate Tm analysis is possible.
本発明の方法は、核酸プローブとターゲット核酸とのハイブリッドを含む溶液の温度を上昇させ、この昇温中に、ハイブリッドの融解により変化するシグナルを測定する測定工程、および、シグナルの変化により核酸プローブとターゲット核酸とのハイブリッドの融解曲線を解析する解析工程を含む融解曲線解析法であり、ハイブリッドのシグナルの測定工程の前に、溶液の温度を、核酸プローブとターゲット核酸とのハイブリッドが形成される温度から、核酸プローブとターゲット核酸が解離する温度まで連続的に上昇させ、次いで、核酸プローブとターゲット核酸とのハイブリッドが形成する温度まで下降させる工程を行うことを特徴とする。 In the method of the present invention, the temperature of a solution containing a hybrid of a nucleic acid probe and a target nucleic acid is increased, and during this temperature increase, a measurement process for measuring a signal that changes due to melting of the hybrid, and a nucleic acid probe by a change in signal Curve analysis method that includes an analysis step of analyzing the melting curve of the hybrid of the target nucleic acid and the target nucleic acid, and before the step of measuring the signal of the hybrid, the temperature of the solution is set, and the hybrid of the nucleic acid probe and the target nucleic acid is formed. The method is characterized in that a step of continuously increasing the temperature from the temperature to a temperature at which the nucleic acid probe and the target nucleic acid are dissociated and then decreasing to a temperature at which a hybrid of the nucleic acid probe and the target nucleic acid is formed is performed.
本発明の方法は、シグナルの測定の前に、核酸プローブとターゲット核酸とのハイブリッドを含む溶液の温度を特定の条件で上昇させてハイブリッドを解離させ、次いで、温度を下降させて核酸プローブとターゲット核酸とのハイブリッドを形成させることの他は、通常のTm解析の方法に従って行うことができる。 In the method of the present invention, prior to signal measurement, the temperature of a solution containing a hybrid of a nucleic acid probe and a target nucleic acid is increased under specific conditions to dissociate the hybrid, and then the temperature is decreased to decrease the temperature of the nucleic acid probe and the target. In addition to forming a hybrid with the nucleic acid, it can be carried out according to the usual Tm analysis method.
核酸プローブは、ターゲット核酸、シグナルの測定方法および解析の目的に応じて適宜設定される。核酸プローブは、通常にはDNAである。 The nucleic acid probe is appropriately set according to the target nucleic acid, the signal measurement method, and the purpose of analysis. The nucleic acid probe is usually DNA.
本発明において、ハイブリッドの融解により変化するシグナルとは、ハイブリッドの融解により生じるまたは大きくなるシグナル、および、ハイブリッドの融解により消失するまたは小さくなるシグナルの両方を包含する。ハイブリッドの融解とは、核酸プローブとターゲット核酸とのハイブリッドが変性して核酸プローブとターゲット核酸に分離することを意味する。シグナルは蛍光であることが好ましい。 In the present invention, the signal changed by melting of the hybrid includes both a signal generated or increased by melting of the hybrid and a signal disappeared or decreased by melting of the hybrid. Hybrid melting means that the hybrid of the nucleic acid probe and the target nucleic acid is denatured and separated into the nucleic acid probe and the target nucleic acid. The signal is preferably fluorescent.
シグナルの測定方法の例としては、蛍光標識した核酸プローブを用いる方法、二本鎖DNAに特異的に結合し、結合したときに蛍光が変化する蛍光試薬を用いる方法等が挙げられる。 Examples of signal measurement methods include a method using a fluorescently labeled nucleic acid probe, a method using a fluorescent reagent that specifically binds to double-stranded DNA and changes fluorescence when bound.
蛍光標識した核酸プローブの例としては、末端が蛍光色素で標識され、ハイブリダイゼーションしたときに蛍光色素の蛍光が減少する核酸プローブ（例えば、特開２００２−１１９２９１号公報参照）等が挙げられる。核酸プローブは一つでなくてもよく、蛍光共鳴エネルギー転移(FRET)効果を生じる２種の蛍光色素でそれぞれ標識され、ターゲット核酸に結合したときにFRET効果が生じるように設定された二つの核酸プローブを用いてもよい。 Examples of fluorescently labeled nucleic acid probes include nucleic acid probes whose ends are labeled with a fluorescent dye and the fluorescence of the fluorescent dye decreases when hybridized (see, for example, JP-A-2002-119291). The number of nucleic acid probes is not limited to two. Two nucleic acids labeled with two fluorescent dyes that produce a fluorescence resonance energy transfer (FRET) effect and set to produce a FRET effect when bound to a target nucleic acid. A probe may be used.
二本鎖DNAに特異的に結合し、結合したときに蛍光が変化する蛍光試薬の例としては、SYBR(商標) Green I色素が挙げられる。 An example of a fluorescent reagent that specifically binds to double-stranded DNA and changes fluorescence when bound is SYBR ™ Green I dye.
蛍光の測定は、蛍光色素に応じた波長の励起光を用い発光波長の光を測定することに行うことができる。 The fluorescence can be measured by measuring light having an emission wavelength using excitation light having a wavelength corresponding to the fluorescent dye.
ターゲット核酸は、通常にはDNAであり、通常の核酸増幅の方法により得られたものであってもよい。核酸増幅の方法としては、PCRポリメラーゼを用いる方法が好ましく、その例としては、PCR、ICAN、LAMP等が挙げられる。PCRポリメラーゼを用いる方法により増幅する場合は、プローブの存在下で増幅を行うことが好ましい。用いる核酸プローブに応じて、増幅の反応条件等を調整することは当業者であれば容易である。 The target nucleic acid is usually DNA, and may be obtained by a normal nucleic acid amplification method. As a method for nucleic acid amplification, a method using PCR polymerase is preferred, and examples thereof include PCR, ICAN, LAMP and the like. When amplifying by a method using PCR polymerase, it is preferable to perform amplification in the presence of a probe. It is easy for those skilled in the art to adjust the amplification reaction conditions and the like according to the nucleic acid probe to be used.
ハイブリッドを含む溶液の組成は、シグナルの測定および融解曲線の解析を妨げない範囲で適宜選択される。すなわち、シグナルの測定、および、核酸プローブとその塩基配列に相補的な配列を有するターゲット核酸とのハイブリダイゼーションが可能であれば特に制限されないが、通常には、一価の陽イオン濃度が1.5〜5 mM、pHが７〜９である。PCR等のDNAポリメラーゼを用いる増幅方法の反応液は、通常、この条件を満たすので、増幅後の反応液をそのままTm解析に用いることができる。 The composition of the solution containing the hybrid is appropriately selected within a range that does not interfere with signal measurement and melting curve analysis. That is, it is not particularly limited as long as signal measurement and hybridization of a nucleic acid probe and a target nucleic acid having a sequence complementary to the base sequence are possible, but usually the monovalent cation concentration is 1.5 to 1.5. 5 mM, pH is 7-9. Since the reaction solution of the amplification method using DNA polymerase such as PCR normally satisfies this condition, the amplified reaction solution can be used as it is for Tm analysis.
測定工程における昇温速度は、通常には、0.1〜1℃／秒である。 The temperature increase rate in the measurement process is usually 0.1 to 1 ° C./second.
本発明において、融解曲線の解析とは、融解温度の決定だけでなく、融解曲線に基づくターゲット核酸の同一性の判別や多型の型の判別も包含する。解析工程は、一塩基多型の部位を有する核酸について、蛍光色素で標識された核酸プローブを用いて、蛍光色素の蛍光を測定することにより融解曲線分析を行い、融解曲線分析の結果に基づいて変異を検出する工程であってもよい。変異を検出する場合には、Tm解析の結果に基づく変異の検出は通常の方法に従って行うことができる。本発明における検出とは、変異の有無の検出の他、変異型DNAの定量、野生型DNAと変異型DNAの割合の測定も包含する。 In the present invention, the analysis of the melting curve includes not only determination of the melting temperature but also determination of the identity of the target nucleic acid based on the melting curve and determination of the polymorphic type. In the analysis step, for a nucleic acid having a single nucleotide polymorphism site, a melting curve analysis is performed by measuring the fluorescence of the fluorescent dye using a nucleic acid probe labeled with the fluorescent dye, and based on the result of the melting curve analysis. It may be a step of detecting a mutation. In the case of detecting a mutation, the detection of the mutation based on the result of Tm analysis can be performed according to a usual method. The detection in the present invention includes not only detection of the presence or absence of mutation, but also quantification of mutant DNA and measurement of the ratio of wild-type DNA and mutant DNA.
本発明の方法では、ハイブリッドのシグナルの測定工程の前に、溶液の温度を、核酸プローブとターゲット核酸とのハイブリッドが形成される温度から、核酸プローブとターゲット核酸が解離する温度まで連続的に上昇させ、次いで、核酸プローブとターゲット核酸とのハイブリッドが形成する温度まで下降させる工程が行われる。 In the method of the present invention, prior to the step of measuring the signal of the hybrid, the temperature of the solution is continuously increased from the temperature at which the hybrid of the nucleic acid probe and the target nucleic acid is formed to the temperature at which the nucleic acid probe and the target nucleic acid are dissociated. Then, a step of lowering to a temperature at which a hybrid of the nucleic acid probe and the target nucleic acid is formed is performed.
温度を連続的に上昇させる工程における昇温速度は、通常には、１〜１００℃／分、好ましくは、６〜６０℃／分である。昇温速度は、測定工程における昇温速度と同じであっても、異なっていてもよい。 The heating rate in the step of continuously increasing the temperature is usually 1 to 100 ° C./min, preferably 6 to 60 ° C./min. The temperature increase rate may be the same as or different from the temperature increase rate in the measurement process.
溶液の温度は、核酸プローブとターゲット核酸とのハイブリッドが形成される温度から、核酸プローブとターゲット核酸が解離する（すなわち、ハイブリッドが融解する）まで上昇させればよい。通常には、室温〜７０℃から９０〜９８℃に上昇させる。 The temperature of the solution may be raised from the temperature at which the nucleic acid probe and the target nucleic acid are hybridized until the nucleic acid probe and the target nucleic acid are dissociated (that is, the hybrid melts). Usually, the temperature is raised from room temperature to 70 ° C to 90 to 98 ° C.
溶液の温度を上昇させるときに、ハイブリッドの融解により変化するシグナルを測定してもよい。すなわち、シグナルの測定工程と同一の工程を行ってもよい。 When raising the temperature of the solution, a signal that changes due to melting of the hybrid may be measured. That is, the same step as the signal measurement step may be performed.
ハイブリッドを形成させる方法は、核酸プローブとターゲット核酸とがアニーリングする限り、特に限定されないが、通常には、４０〜７０℃まで溶液の温度を低下させればよい。 The method for forming the hybrid is not particularly limited as long as the nucleic acid probe and the target nucleic acid are annealed. Usually, the temperature of the solution may be lowered to 40 to 70 ° C.
溶液の温度を連続的に上昇させ、次いで下降させてハイブリッドを形成させる工程は、繰り返しても良い。シグナルの測定工程の前に、溶液の温度を、核酸プローブとターゲット核酸とのハイブリッドが形成される温度から、核酸プローブとターゲット核酸が解離する温度まで連続的に上昇させ、次いで、核酸プローブとターゲット核酸とのハイブリッドが形成する温度まで下降させる工程を行うことにより、シグナルの変化量が大きくなり、従って、高感度なTm解析が可能になる。溶液の温度を連続的に上昇させ、次いでハイブリッドが形成する温度まで下降させる工程として、シグナルの測定工程と同一の工程を行う場合には、必要とする感度が得られるまで、この工程を繰り返すようにしてもよい。 The step of continuously raising the temperature of the solution and then lowering it to form a hybrid may be repeated. Prior to the signal measurement step, the temperature of the solution is continuously increased from the temperature at which the nucleic acid probe and target nucleic acid are hybridized to the temperature at which the nucleic acid probe and target nucleic acid dissociate, and then the nucleic acid probe and target By performing the step of lowering to a temperature at which a hybrid with nucleic acid is formed, the amount of change in the signal increases, and therefore, highly sensitive Tm analysis becomes possible. The temperature of the solution was continuously increased, then a step of lowering to a temperature at which hybridization is formed, in the case of performing the measuring step and the same step of the signal until the sensitivity that needs to obtain, so that the process is repeated It may be.
上記の工程を行うことによりシグナルの変化量が大きくなる理由は、温度を連続的に上昇させてハイブリッドを融解させ、次いで下降させてハイブリッドを形成させることにより、ハイブリッドの融解と形成が、DNAポリメラーゼ反応などのハイブリッド形成に影響を与えるような反応がその途中に生じない条件で行われることになるため、プローブ核酸がターゲット核酸にハイブリダイズしやすくなるためと推定される。 The reason why the amount of change in the signal is increased by performing the above steps is that the temperature is continuously raised to melt the hybrid, and then lowered to form the hybrid. It is presumed that the probe nucleic acid easily hybridizes to the target nucleic acid because the reaction such as a reaction that affects the hybrid formation is performed under conditions that do not occur in the middle of the reaction.
本発明のプログラムは、本発明の方法を実施するためのものであり、核酸プローブとターゲット核酸とのハイブリッドを含む溶液の温度を上昇させ、この昇温中に、ハイブリッドの融解により変化するシグナルを測定し、シグナルの変化により核酸プローブとターゲット核酸とのハイブリッドの融解曲線を解析することを含む融解曲線解析法を行うための、温度の制御が可能な恒温装置の温度を制御する温度制御手段としてコンピューターを機能させるためのプログラムであって、温度制御手段が、ハイブリッドのシグナルの測定のための温度の制御の前に、溶液の温度を、核酸プローブとターゲット核酸とのハイブリッドが形成される温度から、核酸プローブとターゲット核酸が解離する温度まで連続的に上昇させ、次いで、核酸プローブとターゲット核酸とのハイブリッドが形成するまで下降させる温度の制御を行うことを特徴とする。 The program of the present invention is for carrying out the method of the present invention. The temperature of the solution containing the hybrid of the nucleic acid probe and the target nucleic acid is increased, and during this temperature increase, a signal that changes due to melting of the hybrid is generated. As a temperature control means to control the temperature of the thermostat that can control the temperature for measuring and analyzing the melting curve of the hybrid of the nucleic acid probe and target nucleic acid by changing the signal A program for causing a computer to function, wherein the temperature control means determines the temperature of the solution from the temperature at which the hybrid of the nucleic acid probe and the target nucleic acid is formed before controlling the temperature for measurement of the hybrid signal. The nucleic acid probe is continuously raised to a temperature at which the target nucleic acid dissociates, and then the nucleic acid probe And performing temperature control of the lowering until hybrids formed between the target nucleic acid.
温度の制御が可能な恒温装置は、従来の融解曲線解析を行うための装置として知られているものと同様でよく、ハイブリッドの融解によるシグナルを測定できる手段を備えていれば特に限定されない。例えば、ハイブリッドを含む溶液の蛍光を測定できる分光蛍光光度計を備えた恒温装置が挙げられる。 The thermostatic apparatus capable of controlling the temperature may be the same as that known as a conventional apparatus for performing melting curve analysis, and is not particularly limited as long as it has a means capable of measuring a signal due to melting of the hybrid. For example, a thermostat equipped with a spectrofluorometer that can measure fluorescence of a solution containing a hybrid can be mentioned.
ハイブリッドのシグナルの測定工程における温度条件、および、溶液の温度を上昇させ、ハイブリッドを形成させる工程における温度条件は、本発明の方法について説明したのと同様である。 The temperature conditions in the step of measuring the signal of the hybrid and the temperature conditions in the step of raising the temperature of the solution to form a hybrid are the same as described for the method of the present invention.
温度制御手段は、溶液の温度を連続的に上昇させ、次いでハイブリッドを形成させる工程における、昇温速度、到達温度などの温度条件の入力を受ける手段を有していてもよく、その場合には、入力された温度条件に基づいて恒温装置の温度が制御される。 The temperature control means may have means for receiving an input of temperature conditions such as a rate of temperature rise and an ultimate temperature in the step of continuously raising the temperature of the solution and then forming a hybrid. The temperature of the thermostatic device is controlled based on the input temperature condition.
本発明のプログラムは、コンピューターが、ハイブリッドのシグナルの測定のための温度の制御の前に、溶液の温度を、核酸プローブとターゲット核酸とのハイブリッドが形成される温度から、核酸プローブとターゲット核酸が解離する温度まで連続的に上昇させ、次いで、核酸プローブとターゲット核酸とのハイブリッドが形成するまで下降させる温度の制御を行う手段として機能するようにする他は、温度の制御が可能な恒温装置の温度の制御のためのプログラムと同様に作成することができる。 In the program of the present invention, the computer controls the temperature of the solution before measuring the temperature of the hybrid signal from the temperature at which the hybrid of the nucleic acid probe and the target nucleic acid is formed. The thermostatic apparatus is capable of controlling the temperature except that it functions as a means for controlling the temperature continuously raised to the dissociation temperature and then lowered until the hybrid of the nucleic acid probe and the target nucleic acid is formed. Can be created similar to a program for temperature control.
以下に、本発明を実施例により具体的に説明する。 Hereinafter, the present invention will be specifically described by way of examples.
膵ラ氏島アミロイドタンパク質（Islet Amyloid Polypeptide（IAPP））のアミノ酸配列の20位のセリンがグリシンに置換するミスセンス変異（S20G変異）をもたらす塩基の変異（以下、「IAPP S20G変異」ともいう）を含む塩基配列（配列番号１）に基づき、S20G変異を含む部分を増幅できるように表２に示すプライマーを設計した。表１中、位置は、配列番号１に示す塩基配列における塩基番号を示す。 Pancreatic islet amyloid protein (Islet Amyloid Polypeptide (IAPP)) amino acid sequence of the 20th position serine to replace the glycine mutation (S20G mutation) that causes a base mutation (hereinafter also referred to as "IAPP S20G mutation") Based on the nucleotide sequence (SEQ ID NO: 1), the primers shown in Table 2 were designed so that the portion containing the S20G mutation could be amplified. In Table 1, the position indicates the base number in the base sequence shown in SEQ ID NO: 1.
次に、表２に示す、末端部にCを有するプローブを設計した。表２中、位置は、配列番号１に示す塩基配列における塩基番号を示す。また、塩基配列中の大文字は、IAPP S20G変異の部位を示し、3'末端の(P)は、リン酸化されていることを示す。BODIPY(商標) FL及びTAMRA(商標)による標識は、常法に従って行った。 Next, probes having C at the end shown in Table 2 were designed. In Table 2, the position indicates the base number in the base sequence shown in SEQ ID NO: 1. In addition, capital letters in the base sequence indicate the site of IAPP S20G mutation, and (P) at the 3 ′ end indicates phosphorylation. Labeling with BODIPY ™ FL and TAMRA ™ was performed according to conventional methods.
精製ヒトゲノム（２コピー／μｌ）をサンプルとして、Smart Cycler System（Cephied)を用い、以下の条件でPCR及びTm解析を行った。Tm解析における励起波長及び検出波長は、それぞれ450〜495nm及び505〜537 nm(BODIPY FL)、527〜555 nm及び565〜605 nm(TAMRA)であった。 Using the purified human genome (2 copies / μl) as a sample, PCR and Tm analysis were performed using the Smart Cycler System (Cephied) under the following conditions. The excitation wavelength and detection wavelength in Tm analysis were 450 to 495 nm and 505 to 537 nm (BODIPY FL), 527 to 555 nm, and 565 to 605 nm (TAMRA), respectively.
Tm解析は、PCR増幅後の反応液を95℃に加熱し、増幅産物を一本鎖に変性させた後、50℃まで温度を下げてプローブをターゲット核酸（ターゲット)にハイブリダイズさせ、次
に、温度を95℃まで1℃/secの昇温速度で上昇させ、この昇温中の蛍光を測定して行った。さらに、測定後の反応液の温度を50℃まで低下させ、プローブをターゲットにハイブリダイズさせた後、温度を95℃まで1℃/secの昇温速度で上昇させ、この昇温中の蛍光を測定するTm解析の工程を繰り返した。
In Tm analysis, the reaction solution after PCR amplification is heated to 95 ° C, the amplification product is denatured into single strands, then the temperature is lowered to 50 ° C, and the probe is hybridized to the target nucleic acid (target). The temperature was increased to 95 ° C. at a rate of 1 ° C./sec, and the fluorescence during the temperature increase was measured. Furthermore, the temperature of the reaction solution after the measurement is lowered to 50 ° C., the probe is hybridized to the target, and then the temperature is increased to 95 ° C. at a rate of 1 ° C./sec. The process of measuring Tm was repeated.
結果を、図１に示す。図１から明らかなようにTm解析の工程を繰り返すことにより、シグナルが強くなること、すなわち、Tm解析の感度が向上することが明らかになった。 The results are shown in FIG. As is clear from FIG. 1, it became clear that the signal becomes stronger by repeating the process of Tm analysis, that is, the sensitivity of Tm analysis is improved.
なお、図１において縦軸は、蛍光強度の一次導関数の逆符号の値（-dF/dt）、横軸は温度（℃）である。 In FIG. 1, the vertical axis represents the value of the inverse sign of the first derivative of the fluorescence intensity (-dF / dt), and the horizontal axis represents the temperature (° C.).
Claims (4)
ハイブリッドのシグナルの測定の前に、溶液の温度を、核酸プローブとターゲット核酸とのハイブリッドが形成される温度から、核酸プローブとターゲット核酸が解離する温度まで連続的に上昇させ、次いで、核酸プローブとターゲット核酸とのハイブリッドが形成する温度まで下降させる工程を行うことを特徴とする解析法。 The temperature of the solution containing the hybrid of the nucleic acid probe and the target nucleic acid is increased, and during this temperature increase, the signal that changes due to melting of the hybrid is measured, and the melting curve of the hybrid of the nucleic acid probe and target nucleic acid is determined by the change in signal. A melting curve analysis method comprising analyzing,
Prior to the measurement of the hybrid signal, the temperature of the solution is continuously increased from the temperature at which the nucleic acid probe and target nucleic acid are hybridized to the temperature at which the nucleic acid probe and target nucleic acid dissociate, An analysis method comprising a step of lowering to a temperature at which a hybrid with a target nucleic acid is formed.
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