JP3965872B2 - New quantitative polymorphism analysis method - Google Patents

Description

【００５１】
その結果を表１に示した。表１から分かるように、蛍光色素で標識された蛍光消光プローブが標的遺伝子にハイブリダイゼーションしたときに、当該末端部において、当該プローブと標的遺伝子とがハイブリダイゼーションした末端塩基対部分においてＧＣペアーを形成しているか、または末端塩基対部分から１〜３塩基離れて、標的遺伝子の塩基配列にＧ（グアニン）が少なくとも１塩基以上存在するように、当該プローブの塩基配列が設計されていることが好適であることを示している。
【００５２】
実施例２
下記のような塩基配列の標的遺伝子（デオキシリボオリゴヌクレオチド）と本発明の蛍光消光プローブを調製した。標的遺伝子内のＧ及び本発明の核酸プローブ内のＧの数の影響について、前記実施例と同様にして調べ、その結果を表２にまとめた。
【００５３】
名称 標的遺伝子
poly k 5'TATATATATATTTTTGGGGG3'
poly l 5'TATATATATATTTTTTGGGG3'
poly m 5'TATATATATTTTTTTTTGGG3'
poly n 5'TATATATATTTTTTTTTTGG3'
poly o 5'TATATATATTTTTTTTTTTG3'
【００５４】
名称 標的遺伝子
poly p 5'TATATATATATTTTTCCCCC3'
poly q 5'TATATATATATTTTTTCCCC3'
poly r 5'TATATATATTTTTTTTTCCC3'
poly s 5'TATATATATTTTTTTTTTCC3'
poly t 5'TATATATATTTTTTTTTTTC3'
poly u 5'TATATATATTTTTTTTTTTT3'
【００５５】
名称 蛍光消光プローブ
probe k 3'ATATATATATAAAAACCCCC5'-BODIPY FL/C6
probe l 3'ATATATATATAAAAAACCCC5'-BODIPY FL/C6
probe m 3'ATATATATATAAAAAAACCC5'-BODIPY FL/C6
probe n 3'ATATATATATAAAAAAAACC5'-BODIPY FL/C6
probe o 3'ATATATATATAAAAAAAAAC5'-BODIPY FL/C6
【００５６】
名称 蛍光消光プローブ
probe p 3'ATATATATATAAAAAGGGGG5'-BODIPY FL/C6
probe q 3'ATATATATATAAAAAAGGGG5'-BODIPY FL/C6
probe r 3'ATATATATATAAAAAAAGGG5'-BODIPY FL/C6
probe s 3'ATATATATATAAAAAAAAGG5'-BODIPY FL/C6
probe t 3'ATATATATATAAAAAAAAAG5'-BODIPY FL/C6
probe u 3'ATATATATATAAAAAAAAAA5'-BODIPY FL/C6
【００５７】
【表２】

【００５８】
表２から分かるように、蛍光消光プローブが標的遺伝子にハイブリダイゼーションしたときに、当該末端部においてハイブリダイゼーション物の塩基対がＧとＣのペアーを少なくとも一対以上形成するように、当該プローブの塩基配列が設計されていることが好適であることが分かる。
【００５９】
実施例３
下記のような塩基配列の標的遺伝子と蛍光消光プローブを調製した。標的遺伝子内の塩基種および蛍光消光プローブ内の塩基種の影響について、前記実施例と同様にして調べ、その結果を表３にまとめた。
【００６０】
名称 標的遺伝子
poly W 5'CCCCCCTTTTTTTTTTTT 3'
poly X 5'GGGGGGAAAAAAAAAAAA 3'
poly Y 5'TTTTTTCCCCCCCCCCCC 3'
poly Z 5'AAAAAAGGGGGGGGGGGG 3'
【００６１】
名称 蛍光消光プローブ
probe w BODIPY FL/C6-5'AAAAAAAAAGGGGGG 3'
probe x BODIPY FL/C6-5'TTTTTTTTTCCCCCC 3'
probe y BODIPY FL/C6-5'GGGGGGGGGAAAAAA 3'
probe z BODIPY FL/C6-5'CCCCCCCCCTTTTTT 3'
【００６２】
【表３】

表３から、本発明の蛍光消光プローブの蛍光強度の減少にＧが深く関与していることが分かる。
【００６３】
結果として、前記の実施例から、(A)蛍光色素で標識される蛍光消光プローブの末端がＣで構成され、標的遺伝子がハイブリダイゼーションしたとき、ＧＣペアーを形成する場合、(B)蛍光色素で標識される蛍光消光プローブの末端がＣ以外の塩基で構成されているとき、蛍光色素で標識されている個所の塩基と、標的遺伝子の塩基との末端塩基ペアーより、標的遺伝子の３’末端側にＧが少なくとも１個以上存在する場合に、蛍光強度の減少率が大きいことが分かる。
【００６４】
実施例４
本発明の蛍光消光プローブに標識する色素の種類について、前記実施例と同様にして調べた。なお、当該プローブは、前記実施例３の蛍光消光プローブｚを、また、標的遺伝子は前記実施例３の標的遺伝子ｚを用いた。
その結果を、表４に示した。表から分かるように、本発明に用いる蛍光色素として好適なものは、FITC、BODIPY FL、BODIPY FL/C3、6-joe、TMRなどを挙げることができる。
【００６５】
【表４】

【００６６】
実施例５
蛍光色素BODIPY FLで修飾した蛍光消光プローブEu47FおよびEu1392Rの調製
（５−１）蛍光消光プローブEu47Fの合成
（5')CITAACACATGCAAGTCG(3')（I=inosine）の塩基配列をもつデオキシリボオリゴヌクレオチドの５’末端のリン酸基に、実施例１と同じようにしてボデピーFLで標識した蛍光消光プローブEu47Fを合成した。
【００６７】
（５−２）蛍光消光プローブEu1392Rの合成
（5')TTGTACACACCGCCCGTCA(3')の塩基配列をもつデオキシリボオリゴヌクレオチドを用いて、前記（５−１）と同様にして、本発明の蛍光消光プローブEu1392Rの合成した。
【００６８】
実施例６
（６−１）大腸菌JM109株の培養
５３培地（組成：カゼインペプトン（カゼインのトリプシン消化物）、10g；酵母エキス、5g；グルコース、5g；食塩、5g；蒸留水、1000mL）用いて大腸菌JM109株を培養した（培地５０mL／２５０mL容コニカルフラスコ、３７℃、１２時間、振とう培養）。そして、培養液から菌体を集めた（遠心分離１０，０００rpm、５分、蒸留水で２回洗浄）。
【００６９】
（６−２）１６ＳｒＲＮＡのｃＤＮＡの調製
菌体から、SOGENキット（ニッポンジーン社）を用いて全ＲＮＡを本キットのプロトコルに従って抽出した。
その後、BcaBEST^TM RNA PCRキット（宝酒造株式会社）を用い、本キットのプロトコルに従って、前記抽出液について、16sRNAを対象とした増幅と逆転写反応（RT-PCR）を公知の通常の条件で行った。その際、前記の本発明の蛍光消光プローブEu1392Rをプライマーとして用いた。続いて、RNAをRnase Hにより分解し（３０℃、２０分）、16SrRNA遺伝子の純粋なcDNAを得た。cDNA濃度をOliGreen^R ssDNA Quantitationキット（Molecular Probes)を使用して測定した。
【００７０】
実施例７
（７−１）定量的ＰＣＲ、データ解析およびcDNAの検量線の作成
前記cDNA溶液について、本発明の蛍光消光プローブEu47FおよびEu1392Rをプラマーとして用い、リアルタイムモニタリング定量的ＰＣＲ反応を行った。なお、前者はフォワード（forward）型のプライマーとし、後者をリバース（reverseまたはbackward）型のプライマーとした。
リアルタイムモニタリング定量的ＰＣＲ装置として、LightCycler^TM Sytem（ロシュ・ダイアグノスティックス株式会社、ドイツ）を使用し、手順書記載の手順に従って反応を行った。なお、ＤＮＡポリメラーゼとしてTaKaRaTaq ^TM （宝酒造株式会社）を使用した。
【００７１】
ＰＣＲは次のコンポーネントで行った。

なお、cDNAは、図１の注に示される実験区のコピー数で実験を行った。MgCl₂の最終濃度は２mMであった。
【００７２】
反応条件は次の如くであった。

測定条件は次の如くであった。
励起光 ： ５４３ｎｍ
測定蛍光色 ： ５６９ｎｍ
【００７３】
前記の条件でリアルタイムモニタリング定量的ＰＣＲを行って、各サイクルの蛍光強度を実測した。その実測値を本発明のデーター解析方法に従って解析した。すなわち、次の過程でデータを処理した。
（ａ）各サイクルにおける増幅した核酸が蛍光色素で標識された核酸プライマーとハイブリダイズしたときの反応系の蛍光強度値（すなわち核酸伸長反応終了時（７２℃）の蛍光強度値）を、増幅した核酸が核酸プライマーとハイブリダイズしたものが完全に解離したときの反応系の蛍光強度値（すなわち核酸熱変性反応終了時（９６℃）の蛍光強度値）で割る補正演算処理過程、すなわち、実測の蛍光強度値を［数式１］で補正した。
【数８】
ｆ_n＝ｆ_hyb,n／ｆ_den,n ・・・［数式１］
［式中、ｆ_n＝サイクルの蛍光強度の補正値、ｆ_hyb,n＝各サイクルの７２℃の蛍光強度値、ｆ_den,n＝各サイクルの９６℃の蛍光強度値］
【００７４】
（ｂ）各サイクルにおける［数式１］における補正演算処理値を［数式３］に代入し、各サイクルにおける各サンプル間の蛍光消光率を算出する演算処理過程、すなわち、下記の［数式１０］で演算処理する過程、
【数９】
Ｆ_n＝ｆ_n／ｆ₂₅ ・・・［数式１０］
［式中、Ｆ_n＝各サイクルの演算処理値、ｆ_n＝［数式１］で得られた各サイクルの値、ｆ＝［数式１］で得られた値で、サイクル数が２５回目のもの］。
［数式１０］は［数式３］において、ａ＝２５とした場合におけるものである。
【００７５】
（ｃ）前記（ｂ）の過程で得られた各サイクルの演算処理値を［数式６］による蛍光強度の変化率（減少率または消光率）の対数値を演算処理をする過程、すなわち、下記の［数式１１］で演算処理する過程、
【数１０】
ｌｏｇ₁₀｛（１−Ｆ_n）×１００｝ ・・・［数式１１］
［式中、Ｆ_n＝［数式１０］で得られた値］。
［数式１１］は［数式６］において、ｂ＝１０，Ａ＝１００とした場合におけるものである。
【００７６】
上記の結果を図１に示した。
図１は、前記（ａ）、（ｂ）、（ｃ）の過程で計算された値を、サイクル数に対してプロットし、印字したものである。
【００７７】
次に、図１のグラフを基に、次の（ｄ）および（ｅ）の過程で処理した。
（ｄ）前記（ｃ）の過程で処理されたデーターの内、０．２をスレッシュホールド（threshhold）し、その値に達したサイクル数を計算する過程。
（ｅ）前記（ｄ）の過程で計算した値をＸ軸に、反応開始前のコピー数をＹ軸にプロットしたグラフ、すなわち大腸菌cDNAの検量線（図２）を作成する過程。
図２は、本発明の定量的ＰＣＲ方法で得られるデータを、本発明のデータ解析方法、すなわち、（ａ）、（ｂ）、（ｃ）、（ｄ）、（ｅ）の過程で処理した最終結果である。図２から未知コピー数の核酸試料について反応開始前のコピー数を精度よく求めることができることが分かる。
【００７８】
実施例８
（８−１）多型系（複合微生物系）の構築
（表５に示した１０種類の細菌菌株をDSMZ（Deutshe Sammlung von Mikroorganismen und Zellkulturen GmbH）から購入し、前記の５３培地を用いて各々の菌株ついて別個に培養した。培養条件は前記大腸菌の場合と同様である。各々の培養液から菌体を集めた（遠心分離１０，０００ｒｐｍ、１０分、蒸留水で２回洗浄）。各々の菌体について、前記と同様にしてＳＯＧＥＮキット（ニッポンジーン社）を用いて全RNAを抽出した。
【００７９】

【００８０】
１：Paracoccus pantotrophus
２：Sphingomonas natatoria
３：Bdellovibrio stolpii
４：Microbacterium imperiale
５：Pseudomonas fluorescens
６：Agromyces medislanum
７：Cellulomonas cellulans
８：Brevibacterium liquefaciens
９：Leminorella grimontii
１０：Rhodococcus luteus
【００８１】
その後、前記の大腸菌の場合と同様にして、それぞれの菌株の16SrRNA遺伝子の純粋なcDNAを得た。得られた１０菌株各々のcDNA濃度を前記の大腸菌の場合と同様にして測定した。cDNA濃度の判明した溶液について、蒸留水にて３００，０００copy/μLとなるように希釈した。１０菌株分について、希釈液を当量づつ混合したものを複合微生物系すなわち多型系（以下、多型系という。）とした。この多型系には、１０菌株分のｃＤＮＡがそれぞれ３００，０００copy/μLの濃度で含まれているので、全体として、３，０００，０００copy/μLの濃度cDNAが含有していることになる。
【００８２】
（８−２）リアルタイムモニタリング定量的ＰＣＲ
前記多型系のcDNAについて、本発明の蛍光消光プローブEu47FおよびEu1392Rを菌株共通のプラマーとして用いて、前記大腸菌と同様にしてリアルタイムモニタリング定量的ＰＣＲを行った。
多型系のサンプルを、絶対量で３００，０００copy/２０μL（反応液全体２０μL）となるように反応液に添加した。多型系のリアルタイムモニタリング定量的ＰＣＲは、蛍光強度の減少が観察され、かつ遺伝子の指数関数的増幅期である２２サイクル数で反応を停止させた（図１参照）。スレッシュホールドを、log Rn（蛍光消光率）＝０．２と設定したときの、多型系について行ったリアルタイムモニタリング定量的ＰＣＲの反応液のcDNAのコピー数は２８８，０００コピーであった（図２参照）。初期添加量すなわち理論値は３００，０００コピーであるから、本発明の方法によって作成された検量線は良好な定量性を示すことが確認された。
【００８３】
実施例９
多型解析
（９−１）T-RFLPによる解析
前記のようにしてＰＣＲ反応を行った後、増幅産物をカラム（MicroconPCR、Millipore Corporation Bedford、MA、USA）を用いて精製した。精製物を制限酵素Hha1（認識部位：GCG/C、／＝切断個所）でO/N（一晩）処理した。処理終了後、切断断片のみをカラム（Microcon及びMicropure-EZ、Millipore Corporation Bedford、MA、USA）で精製した。制限酵素処理後の各菌株のｃＤＮＡ断片の大きさは、表５に示した。
カラム精製を施したcDNA溶液について、加熱変性処理を行った後、シーケンサー（ABI PRISMTH 310、PE Applied Biosystems）にてT-RFLP解析を行った。そのピークパターンを図３に示した。各ピークを濃度が既知である標準BODIPY FL修飾断片を用いて定量した。各ピークのモル構成率を求めた結果、すべてのモル構成率は、９．４〜１０．８の範囲に収まっており、ＰＣＲ増幅効率の極端な差異認められなかった（表５参照）。定量的ＰＣＲで求めてた全ｃＤＮＡのコピー数にモル構成率を掛け、それぞれの菌株の初期のcDNAのコピー数を求めた（表５参照）。定量により求めたコピー数／初期添加コピーは０．８９〜１．０４（表５参照）であった。よって、本方法により多型系における多型の初期コピーを正確に定量できることが判明した。
【００８４】
【発明の効果】
本発明の定量的多型解析方法は、上記のような構成であるために次のような効果を有する。
１）標的遺伝子の量及び該遺伝子の多型の構成比の測定を簡便、迅速かつ定量性よく行うことができる。
２）本発明の定量的ＰＣＲ方法の一つは新規なものであり、かつ定量性が優れたものである。
３）定量的ＰＣＲ方法で得られたデータを解析する解析方法の一つも新規な方法であり、ＰＣＲ反応前の初期の遺伝子のコピー数を正確に求めることができる。
４）本発明の新規な定量的ＰＣＲ方法においては、増幅核酸は蛍光色素で標識されている。それで、多型の解析では蛍光色素をマーカとして分析できる。
５）２）〜４）の結果、特に定量性の優れた多型解析方法になる。
【図面の簡単な説明】
【図１】 本発明の定量的ＰＣＲ方法を用いた１６ＳｒＲＮＡ遺伝子（ｃＤＮＡ）の増幅曲線
実線：大腸菌のｃＤＮＡ；点線：多型系のｃＤＮＡ
１０²、１０³、１０⁴、１０⁵、１０⁶：コピー数を表示する。
【図２】 本発明のデータ解析方法によって作成されたｃＤＮＡの検量線
ａ：２８８０００コピー
【図３】 本発明の多型系のT-RFLPの解析パターン
ｂｐ：塩基数（base pair）[0001]
BACKGROUND OF THE INVENTION
  The present invention provides a novel quantitative polymorphism analysis method,Real-time monitoring quantitative PCR method used therefor and fluorescence quenching probe used thereforAbout.
[0002]
[Prior art]
The method of amplifying a target gene by PCR method (protein / nucleic acid / enzyme; Volume 35, No. 17, 1990, Kyoritsu Shuppan Co., Ltd.) and polymorphism analysis of the target gene has been greatly improved. Widely used in various fields such as medicine (Experimental Medicine, Vol. 15, No. 7, 1997, Yodosha). And elucidation from genes of various diseases, especially diseases related to immunity, has been achieved.
[0003]
However, in the conventional polymorphism analysis method, since it was made by using non-quantitative PCR, only one target gene is amplified among genes having similar sequences (for example, extracted from activated sludge). (It amplifies only the 16S rRNA gene (DNA) sequence of one specific microbial species from the total DNA obtained), so analysis of the initial gene amount and polymorphic composition ratio before amplifying the target gene I could not do it.
[0004]
[Problems to be solved by the invention]
In view of the above situation, an object of the present invention is to provide a novel polymorphism analysis method for simply and rapidly measuring the amount of a target gene before amplification and the polymorphism ratio of the gene, and a novel quantification used in the method. PCR method, fluorescence quenching probe used in the quantitative PCR method, novel analysis method for analyzing data obtained by the quantitative PCR method, reagent kit for quantitative PCR method, reagent kit for quantitative polymorphism analysis method, A computer-readable recording medium for data analysis of a PCR method that records a program for causing a computer to execute a method for analyzing data obtained by a quantitative PCR method, and analysis of data obtained by a quantitative polymorphism analysis method Computer-readable recording medium having recorded program of procedure for causing computer to execute method to perform, quantitative PC And to provide an analysis device for analysis apparatus and quantitative polymorphism analysis for the process.
[0005]
[Means for Solving the Problems]
  As a result of diligent efforts to solve the above problems, the present inventors haveReal-time monitoringquantitativePCR method (Gene amplification method)After the target gene is amplified using, the polymorphism of the target gene is analyzed, so that the amount of the target gene before amplification and the composition ratio of the polymorphism of the gene can be measured easily, quickly and quantitatively.LineI got the knowledge that I can. The present invention has been completed based on such findings.
[0006]
  That is, the present invention
1) A target gene is amplified by a real-time monitoring quantitative PCR method, the initial concentration of the target gene amount is measured, and T-RFLP (terminal restriction fragment length polymorphism), RFLP (restriction) fragment length polymorphism) method, SSCP (single strand conformation) method, or CFLP (cleavase fragment length polymorphism) method to analyze the amount of the initial target gene and the polymorphism composition ratio or the amount of the gene. MeasureDefiniteQuantitative polymorphism analysis methodThe quantitative polymorphism analysis method, characterized in that the real-time monitoring quantitative PCR method is a reaction using the following fluorescence quenching probe as a primer to measure the amount of decrease in the emission of the fluorescent dye,
  Fluorescence quenching probe: at its 5 ′ end or at its 5 ′ end phosphate group 6-joe , BODIPY TMR , BODIPY FL , BODIPY FL / C3 , BODIPY FL / C6 , Alexa 488 , Alexa 532 When the probe labeled with any of the fluorescent dyes is hybridized to the target gene at the end, the base sequence of the target gene is separated from the terminal base pair part of the target gene hybridized to the probe by 1 to 3 bases A fluorescence quenching probe in which the fluorescent dye reduces the fluorescence intensity when the probe base sequence is designed so that at least one base of G (guanine) is present in the probe and the probe hybridizes to the target gene.
Or
2) The real-time monitoring quantitative PCR method described above in 1), wherein the amplification reaction of the target gene is stopped at the exponential amplification phase.FixedQuantitative polymorphism analysis method, or
3) The real-time monitoring quantitative PCR method comprises:following1) or 2), wherein the reaction is carried out using the fluorescence quenching probe as a primer to measure the amount of decrease in the emission of the fluorescent dye.FixedQuantitative polymorphism analysis method,
  Fluorescence quenching probe: at its 5 ′ end or at its 5 ′ end phosphate group 6-joe , BODIPY TMR , BODIPY FL , BODIPY FL / C3 , BODIPY FL / C6 , Alexa 488 , Alexa 532 When the probe is hybridized to the target gene, the base pair of the hybrid complex of the probe and the target gene is at least G (guanine) and C (cytosine) at the terminal. A fluorescence quenching probe in which the fluorescent dye decreases the fluorescence intensity when the base sequence of the probe is designed to form a pair of and the probe hybridizes to a target gene.
I will provide a.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
Next, the present invention will be described in more detail with reference to preferred embodiments.
First, terms used in the present invention are defined as follows.
“Target gene” refers to a nucleic acid or gene that is the target of measurement / quantification or quantitative detection of an abundance. The gene consists of DNA, RNA, oligodeoxyribonucleotide, and oligoribonucleotide. In addition, the gene may be a mixture of these various genes, such as proteins, high molecular compounds such as polysaccharides, amino acids, saccharides, nucleotides, bases, vitamins, inorganic compounds, etc. Low molecular weight compounds may be mixed. Moreover, you may exist in a cell. That is, there are no special restrictions. The cells may be eukaryotic cells or prokaryotic cells. Moreover, various cells may be mixed. Regardless of purification. Moreover, the magnitude | size of a density | concentration does not ask | require. For example, a specific gene for analysis in a complex microbial system or a symbiotic microbial system. When purification is necessary, it can be performed by a conventionally known method. For example, it can be performed using a commercially available purification kit.
[0008]
“PCR” is a polymerase chain reaction, and is currently a reaction commonly used in molecular biology, molecular cytology, genetic engineering, and the like.
The “quantitative PCR method” is a method for measuring or detecting the abundance of a target gene before PCR by performing PCR.
A “fluorescence quenching probe” is a probe designed to reduce the fluorescence intensity when hybridized to a target gene with a nucleic acid probe labeled with a fluorescent dye.
[0009]
“Primers, nucleic acid probes and hybridization, hybridization in PCR” have the same meaning as currently used in molecular biology, genetic engineering, and the like.
[0010]
“Polymorphism” refers to biological polymorphisms. In the present invention, it is particularly a polymorphism of a gene (RNA, DNA, gene) that causes the polymorphism. The meaning is currently used in molecular biology.
“Polymorphism analysis” is to analyze and analyze what kind of polymorphism a gene has.
[0011]
Currently, polymorphism analysis methods include SSOP (sequence specific oligonucleotide probe) method, RFLP (restriction fragment length polymorphism) method, T-RFLP (terminal restriction fragment length polymorphism) method, SSCP (single strand conformation) method, MPH Method, CFLP (cleavase fragment length polymorphism) method, SSP (ssequences specific primer) method, PHFA (preferential homoduplex formation formation assay) method, SBT (sequence based typing) method (PCR method, usage guide, Chugaisha, 1998) 35; No. 17, 1990, Kyoritsu Publishing Co., Ltd .; Experimental Medicine, Vol. 15, No. 7 (extra number, 1997), etc. Although all methods used for the analysis can be used, in particular, the T-RFLP method or the CFLP method can be preferably used.
[0012]
Hereinafter, the present invention will be specifically described step by step.
The first feature of the present invention is a quantitative gene amplification method. As the quantitative gene amplification method, any method having quantitativeness can be adopted. For example, a PCR method can be suitably employed. Among them, the quantitative PCR method or the real-time monitoring quantitative PCR method is a more preferable method. Among these, the novel real-time monitoring quantitative PCR method of the present invention using a fluorescence quenching probe can be suitably employed.
Conventionally known quantitative PCR methods include, for example, RT-PCR, RNA-primed PCR, Stretch PCR, reverse PCR, PCR using Alu sequences, multiplex PCR, PCR using mixed primers, PCR using PNA, etc. The method of etc. can be mentioned.
[0013]
These conventionally known quantitative PCR methods use dATP, dGTP, dCTP and dTTP or dUTP, a target gene (DNA or RNA), Taq polymerase, a primer, and a nucleic acid probe or intercalator labeled with a fluorescent dye. In the presence of, the target gene is amplified while repeating the temperature at low and high temperatures, and the increase in the emission of the fluorescent dye in the amplification process is monitored in real time (Experimental Medicine, Vol. 15, No. 7, 46-51). Page, 1997, Yodosha).
[0014]
The quantitative PCR method using the fluorescence quenching probe of the present invention is a method using a probe labeled with a fluorescent dye, and a probe designed to reduce or quench fluorescence when the probe is hybridized with a target gene. Is a quantitative PCR method using
For example, the fluorescence quenching probe is labeled with a fluorescent dye at its end, and when the probe hybridizes to the target gene at the end, 1 to 3 from the terminal base pair portion of the target gene hybridized to the probe. The base sequence of the probe is designed so that there is at least one base G (guanine) in the base sequence of the target gene, and when the probe hybridizes to the target target gene, the fluorescent dye is fluorescent. Those that reduce the strength are preferred. Among these, those in which the fluorescence quenching probe is labeled with a fluorescent dye at the 3 'end or 5' end are more preferable.
[0015]
The fluorescence quenching probe is labeled with a fluorescent dye at its end, and when the probe is hybridized to the target gene, the base pair of the hybrid complex of the probe and target gene is at least G (guanine) at the end. That the base sequence of the probe is designed to form a C (cytosine) pair (GC base pair) and the probe reduces the fluorescence intensity when the probe is hybridized to the target gene Is preferred. Among them, those in which the fluorescence quenching probe is labeled with a fluorescent dye at the 3 'end or the 5' end are more preferable.
[0016]
In this case, if the 5 ′ or 3 ′ end cannot be designed as G or C from the base sequence of the target gene, 5′-guanylic acid or 5 ′ at the 5 ′ end of the oligonucleotide that is a primer designed from the base sequence of the target gene Even when 5′-cytidylic acid is added, the object of the present invention can be preferably achieved. Therefore, the fluorescence quenching probe of the present invention is a probe designed from the base sequence of a target gene, and 5′-guanylic acid or 5′- at the 3 ′ or 5 ′ end, preferably 5 ′ end of the probe. It is defined as including a probe formed by adding cytidylic acid.
[0017]
In the present invention, a fluorescent dye generally labeled with a nucleic acid probe and used for nucleic acid measurement / detection can be conveniently used. However, when a nucleic acid probe labeled with a fluorescent dye is hybridized to a target gene, As the fluorescent dye labeled with, a fluorescent dye whose emission is reduced, that is, a quenching substance is preferably used. For example, fluorescein or derivatives thereof (for example, fluorescein isothiocyanate (FITC) or derivatives thereof, such as Alexa 488, Alexa 532, cy3, cy5, EDANS (5- (2'-aminoethyl) amino- 1-naphthalene sulfonic acid, rhodamine 6G (R6G) or derivatives thereof (eg, teramethylrhodamine (TMR), tetramethylrhodamine isothiocyanate (TMRITC), x-rhodamine (x- rhodamine, Texas red, BODIPY FL (trade name; manufactured by Molecular Probes, USA), BODIPY FL / C3 (trade name; molecular probes) Manufactured by the United States), BODIPY FL / C6 (trade name; manufactured by Molecular Probes, USA), BODIPY 5-FAM (trade name; manufactured by Molecular Probes, USA), BODIPY TMR (trade name; manufactured by Molecular Probes, USA), or a derivative thereof ( For example, BODIPY TR (trade name; manufactured by Molecular Probes, USA), BODIPY R6G (trade name; manufactured by Molecular Probes, USA), BODIPY 564 (trade name; manufactured by Molecular Probes, USA), BODIPY 581 (trade name; manufactured by Molecular Probes, USA), and the like. FITC, EDANS, 6-joe, TMR, Alexa 488, Alexa 532, BODIPY FL / C3 (trade name; manufactured by Molecular Probes, USA), BODIPY FL / C6 (trade name; manufactured by Molecular Probes, USA) and the like are suitable, and FITC, TMR, 6-jeo, BODIPY FL / C3 (trade name; molecular probe (Molecular Probes) Probes), USA, and BODIPY FL / C6 (trade name; manufactured by Molecular Probes, USA) can be mentioned as more preferable examples.
[0018]
The primer labeled with the fluorescent dye used in the present invention is composed of oligodeoxyribonucleotide or oligoribonucleotide, and the number of bases thereof is 5 to 50, preferably 10 to 25, particularly preferably 15 to 20. Any base sequence that specifically hybridizes to the target gene may be used.
[0019]
The oligonucleotide of the fluorescence quenching probe labeled with the fluorescent dye of the present invention can be produced by an ordinary general oligonucleotide production method. Can be prepared by chemical synthesis methods or by microbial methods using plasmid vectors or phage vectors (Tetrahedron letters, 22, 1859-1862, 1981; Nucleic acids Research, 14, 6227-6245, 1986) . It is preferable to use a commercially available nucleic acid synthesizer (for example, ABI394 (manufactured by Perkin Elmer)).
When a fluorescence quenching probe is used as a primer for a quantitative PCR method, it may be designed to be either a forward type, a reverse type, or a backward type.
[0020]
The fluorescent dye can be labeled on the oligonucleotide by a conventionally known labeling method (Nature Biotechnology, 14, 303-308, 1996; Applied and Environmental Microbiology, 63, 1143-1147, 1997). Year; Nucleic acids Research, 24, 4532-4535, 1996). For example, when a fluorescent dye molecule is bonded to the 5 'end, first, the 5' end is dephosphorized according to a conventional method, and-(CH as a spacer or linker is added to the OH group at the 5'-position carbon of ribose.₂)_n-SH,-(CH₂)_n-NH₂And so on. These spacers or linkers may be introduced into the phosphate group at the 5 'or 3' position of ribose. The dye of the present invention is bound to these spacers or linkers.
[0021]
These introducers are commercially available and may be purchased commercially (Midland Certified Reagent Company). In this case, n is 3 to 12, preferably 6 to 10. The spacer or linker can be synthesized by binding a reactive fluorescent dye or a derivative thereof to the marker group or amino group. The oligonucleotide labeled with a fluorescent dye synthesized in this way can be purified by chromatography such as reverse phase to obtain the fluorescence quenching probe of the present invention.
[0022]
By the quantitative PCR method using the above-described fluorescence quenching probe of the present invention, a target gene can be amplified in a short time, simply and specifically. When a fluorescence quenching probe labeled with a fluorescent dye at the 5 'end is used, the target gene labeled with the fluorescent dye at the 5' end is amplified. In this case, it is preferable to employ a quantitative PCR method (Experimental Medicine, Vol. 15, No. 7, 1997, Yodosha)
[0023]
As the thermal cycler to be used, various commercially available devices can be used conveniently. It does not matter whether real-time monitoring is possible. For example, ABI PRISM^TM 7700 Sequence Detection System (SDS 7700) (Perkin Elmer Applied Biosytems, USA), LightCycler^TMSytem (Roche Diagnostics Inc., Germany) can be mentioned as a particularly suitable one.
[0024]
Gene amplification can be achieved under conventionally known amplification reaction conditions. Amplification is preferably performed up to the amplification level generally used. In the amplification process of the target gene, the fluorescence intensity value is measured with a fluorometer. The amount of change is proportional to the amount of amplified gene. When the amount of change in fluorescence intensity is plotted as a function of time (number of cycles in the case of PCR method) on a normal graph, it becomes a sigmoid (sigmoid) curve, and when plotted on an edge logarithmic graph, it is similar to the exponential function at first. The curve increases linearly and then reaches a gentle plateau.
The degree of amplification of the target gene to improve the quantification of the initial gene amount before the start of PCR, that is, the timing of stopping the gene amplification reaction depends on the purpose of the polymorphism analysis and is not particularly limited. . That is, when analyzing only the polymorphic preferential polymorphism, it is preferable to amplify the target gene from an arbitrary time until the plateau is reached after the fluorescence change is observed. Most preferably, the reaction is stopped at the exponential amplification period (before reaching the midpoint of the sigmoid curve (the point at which the differential value of the curve reaches 0)). However, when analyzing all the polymorphisms included in the polymorphism system, trials and errors are performed several times, the amplification degree considered to be the best is obtained, and all genes showing polymorphism in the reaction system can be observed. It is better to amplify to the extent. In addition, a method of dividing the amplification into a plurality of times, that is, performing an experiment with a plurality of amplification degrees and comprehensively analyzing the results is also suitably employed. This is because minor polymorphisms draw sigmoid curves with large lags (delays) in time.
[0025]
When the fluorescent quenching probe of the present invention is used as a primer and a quantitative PCR method, particularly a real-time monitoring quantitative PCR method, is performed, the fluorescence quenching probe as a primer is used from one to the next for amplification of a target gene, The target gene labeled at the 5 ′ end with a fluorescent dye is amplified. The amplified target genes are hybridized with target genes corresponding to each other. When hybridized, the fluorescence intensity value decreases. Therefore, the amplification reaction may be performed up to the best amplification degree in the same manner as described above by controlling the amount of decrease in fluorescence intensity. In the quantitative PCR method, the reaction can be carried out under the same reaction conditions as those in the normal PCR method. Thus, the target gene can be amplified in a reaction system in which the Mg ion concentration is low (1-2 mM) or a conventionally known high concentration (2-4 mM).
[0026]
It is preferable to prepare a standard curve of a gene using a standard gene before the amplification reaction of the target gene. For example, the case where the above-mentioned fluorescence quenching probe of the present invention is used as a primer and the real-time monitoring quantitative PCR method is performed will be described.
When the amount of decrease in fluorescence intensity is plotted on a normal graph as a function of the number of cycles, an S-shaped (sigmoid) curve is obtained. The number of cycles at the time of the greatest decrease rate and the initial copy number of the target gene amount (copy number before starting PCR), that is, the initial target gene has an exponential relationship. If a standard straight line of the relationship between the number of cycles and the copy number at these time points is prepared, the initial copy number of the target gene of the unknown sample, that is, the initial target gene amount can be determined.
The quantitative PCR method using the fluorescence quenching probe is a novel method developed by the present inventors.
[0027]
The second feature of the present invention is a novel analysis method for analyzing data obtained by the quantitative PCR method. The analysis method of the present invention is currently most suitable for measuring the initial target gene amount as accurately as possible.
[0028]
First, a data analysis method will be described.
The method consists of three features.
The first feature is the reaction system when the amplified gene in each cycle is bound to the fluorescent dye, or when the amplified gene is hybridized with the fluorescence quenching probe, that is, when a hybrid complex is formed (for example, the end of the annealing reaction). Fluorescence intensity value of the reaction system at the time of the reaction, the reaction system at the time of the nucleic acid extension reaction proceeding or at the end thereof, the combined one in each cycle, or the reaction system when the hybrid complex is dissociated (for example, This is an arithmetic processing step that corrects based on the fluorescence intensity values of the reaction system at the end of the denaturation reaction and the reaction system before the start of the annealing reaction, ie, a correction arithmetic processing step.
[0029]
The correction calculation process in the correction calculation process may be any process that meets the object of the present invention. Specifically, the process according to the following [Formula 1] or [Formula 2] Can be exemplified.
[Expression 1]
f_n= F_{hyb, n}/ F_{den, n}      ... [Formula 1]
[Expression 2]
f_n= F_{den, n}/ F_{hyb, n}      ... [Formula 2]
[Where:
f_n: Correction calculation processing value in n cycles calculated by [Equation 1] or [Equation 2],
f_{hyb, n}: The fluorescence intensity value of the reaction system when the amplified gene binds to the fluorescent dye in the n cycle or when the amplified gene forms a hybrid complex,
f_{den, n}: Fluorescence intensity value of the reaction system when the bound or hybrid complex is dissociated in n cycles].
This process includes a process of plotting the correction calculation processing value or the value obtained in this process on a graph with respect to each cycle number, and displaying and / or printing on a computer display.
[0030]
The second feature is that the correction calculation processing value according to [Equation 1] or [Equation 2] in each cycle is substituted into the following [Equation 3] or [Equation 4], and the fluorescence change rate between samples, that is, the fluorescence quenching rate Is a data analysis process of calculating and comparing them.
[0031]
[Equation 3]
F_n= F_n/ F_a      ... [Formula 3]
[Expression 4]
F_n= F_a/ F_n      ... [Formula 4]
[Where:
F_n: In n cycles, the fluorescence change rate calculated by [Equation 3] or [Equation 4], that is, the fluorescence quenching rate,
f_n: Correction calculation processing value according to [Formula 1] or [Formula 2]
f_a: Correction calculation processing value according to [Formula 1] or [Formula 2], f_nAny number of cycles before the change is observed, but typically 10 to 40 cycles, preferably 15 to 30 cycles, more preferably 20 to 30 cycles. Adopted. ].
In this process, the calculated value, the comparison value or the value obtained in this process is plotted on a graph with respect to each cycle number, and displayed and / or printed on a computer display. , [Numerical expression 1] or [Numerical expression 2] may be included or not included in the correction calculation process value.
[0032]
The third feature is: i) Calculation by [Formula 5], [Formula 6] or [Formula 7] using the data of the fluorescence change rate calculated by [Formula 3] or [Formula 4], that is, the fluorescence quenching rate. Process,
[Equation 5]
log_b(F_n), Ln (F_n) [Formula 5]
[Formula 6]
log_b{(1-F_n) × A}, ln {(1-F_n) × b} [Formula 6]
[Expression 7]
log_b{(F_n−1) × A}, ln {(F_n−1) × A} [Formula 7]
[0033]
[Where:
A, b: Any numerical value, preferably an integer value, more preferably a natural number. When A = 100 and b = 10, {(F_n−1) × A} represents a percentage (%).
F_n: Fluorescence change rate in n cycles calculated by [Equation 3] or [Equation 4], ie, fluorescence quenching rate],
[0034]
ii) an arithmetic processing step for obtaining the number of cycles in which the arithmetic processing value of i) has reached a certain value;
iii) a calculation process for calculating a relational expression between the number of cycles in a nucleic acid sample of a known concentration and the copy number of the target gene at the start of the reaction,
iv) a process of calculating the target gene copy number at the start of PCR in an unknown sample,
Is a data analysis method. A process consisting of i) → ii) → iii) → iv) is preferable.
[0035]
In each of the above steps i) to iv), a calculation process value obtained in each step or the value is plotted on a graph with respect to each cycle number, and displayed and / or printed on a computer display. It may be included.
It should be noted that the correction calculation processing value according to [Formula 1] or [Formula 2] and the calculation processing value according to [Formula 3] or [Formula 4] are plotted on a graph for each cycle number and displayed on a computer display. These processes may be added as necessary because they may or may not be printed.
[0036]
The present invention also provides a computer-readable recording medium in which a reagent kit used in the quantitative PCR method and a program for causing a computer to execute the data analysis method are recorded.
In addition, the present invention is a data analysis apparatus characterized by having means for performing the data analysis method.
[0037]
The third feature of the present invention is a method for analyzing polymorphisms of genes amplified by the quantitative PCR method of the present invention as described above.
Therefore, the polymorphism analysis method will be specifically described. Among various methods of polymorphism analysis, the T-RFLP method can be suitably used in the present invention. Therefore, as one example of the present invention, a gene is amplified and the initial gene amount before PCR is measured by a quantitative PCR method using a fluorescence quenching probe as a primer, particularly a real-time monitoring quantitative PCR method. The amplification product is described as a method for analyzing polymorphisms by the T-RFLP method. The gene amplified using a fluorescence quenching probe as a primer is labeled with the fluorescent dye of the present invention at the 5 'end.
(1) First, the amplification product is digested with a restriction enzyme. At this time, any currently known restriction enzyme can be used.Bso FI,Hha I,Hph I,Mnl I,Rca I,Alu I,Msp I can be mentioned. Among these, preferred are:Hha I,Alu I,Msp The most preferable ones such as I areHha I. The digestion reaction conditions may be normal conditions for currently known genes. For example, as a restriction enzymeHha When I is selected, the reaction is performed at a restriction enzyme concentration of 10 units at 37 ° C. for 6 hours.
[0038]
(2) The gene fragment digested as described above is preferably heat-denatured to be single-stranded. This modification treatment can also be performed under known normal conditions. For example, after treatment at 97 ° C. for 5 minutes, it is cooled in ice.
[0039]
(3) Analysis and analysis of gene fragments
In the polymorphism analysis method of the present invention, only a gene fragment labeled with a fluorescent dye is analyzed and analyzed by an electrophoresis method, an HPLC method, a sequencer method, or the like.
That is, each band and each peak are detected by the fluorescence intensity value. Detection can be performed using conventional analytical instruments currently on the market. Examples include ABI 373A (ABI sequencer), ABI377 (ABI sequencer), Biofocus 3000 (Biorat).
[0040]
In the present invention, the appearance of a plurality of bands or a plurality of peaks in the analysis means that a polymorphism exists. In the case of a single band or a single peak, no polymorphism exists. The ratio of the fluorescence intensity values of each band or each peak is the polymorph abundance ratio. In the quantitative PCR method of the present invention, since the amount of the target gene before PCR is measured, the initial abundance of the polymorphism can be obtained by multiplying the measured value by the ratio of the polymorphism. become.
With respect to polymorphisms, a method for generating data in this way can be realized only by using a quantitative PCR method using the fluorescence quenching probe of the present invention.
In addition, a reagent kit for quantitative polymorphism analysis that is convenient can be obtained by containing or attaching a reagent kit for the quantitative PCR method.
Further, the real-time monitoring quantitative PCR data analysis method is recorded on a computer-readable recording medium recording a program for causing a computer to execute the method for analyzing data obtained by the polymorphism analysis method. By recording the program to be executed together, the computer-readable quantitative polymorphism analysis method becomes a more convenient recording medium for data analysis.
In addition, in a polymorphism analyzer having means for a quantitative polymorphism analysis method, a data analysis apparatus for the PCR method is provided together with the apparatus, thereby providing a convenient polymorphism analyzer.
[0041]
【Example】
Next, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by this embodiment.
Example 1
The base selectivity of the target gene of the fluorescence quenching probe of the present invention having the fluorescence quenching phenomenon, that is, the base specificity was examined. Ten types of poly a to j of the target gene (deoxyribooligonucleotide) (30 mer) shown below were prepared with a DNA synthesizer ABI 394 (Perkin Elmer, USA).
[0042]
Furthermore, the following fluorescence quenching probe of the present invention labeled with bodepy FL was prepared at the 5 'end of the deoxyribooligonucleotide corresponding to the above target gene.
To the phosphate group at the 5 'end of the deoxyribooligonucleotide,-(CH₂)₆-NH₂The product was purchased from Medland Certified Reagent Company (USA). In addition to the Fluo Reporter Kits F-6082 (BODIPY FL propionic acid succinidyl esters) from Molecular Probes, the compounds can be used as amine derivatives of oligonucleotides. Purchased a kit containing a reagent to be bound to. The kit was allowed to act on the purchased deoxyribooligonucleotide to synthesize the fluorescence quenching probes probe a to d and f to h of the present invention labeled with the following Bodepy FL. Then, the degree of fluorescence intensity (that is, the extent of quenching) when hybridized with the corresponding target gene was examined under the following conditions to examine the specificity of the probe of the present invention.
[0043]
The synthesis product was purified as follows.
The synthesized product was dried to obtain a dried product. 0.5M Na₂CO_Three/ NaHCO_ThreeDissolved in buffer (pH 9.0). The lysate was subjected to gel filtration with a NAP-25 column (Pharmacia) to remove unreacted substances. Further, reverse phase HPLC (B gradient: 15 to 65%, 25 minutes) was performed under the following conditions. And the main peak which eluted was fractionated. The fraction obtained was freeze-dried to obtain the desired product in a yield of 50% from 2 mM of the first oligonucleotide raw material.
[0044]
Reversed phase chromatography conditions:
Elution solvent A: 0.05N TEAA 5% CH_ThreeCN
Elution solvent B (for gradient): 0.05N TEAA 40% CH_ThreeCN
Column: CAPCELL PAK C18; 6 x 250mm
Elution rate: 1.0ml / min
Temperature: 40 ° C
Detection: 254nm
[0045]
Name Target gene
poly a 5'ATATATATTTTTTTTGTTTTTTTTTTTTTT3 '
poly b 5'ATATATATTTTTTTTTGTTTTTTTTTTTTT3 '
poly c 5'ATATATATTTTTTTTTTGTTTTTTTTTTTT3 '
poly d 5'ATATATATTTTTTTTTTTGTTTTTTTTTTT3 '
poly e 5'ATATATATTTTTTTTTTTTGTTTTTTTTTT3 '
[0046]
Name Target gene
poly f 5'ATATATATTTTTTTTCTTTTTTTTTTTTTT3 '
poly g 5'ATATATATTTTTTTTTCTTTTTTTTTTTTT3 '
poly h 5'ATATATATTTTTTTTTTCTTTTTTTTTTTT3 '
poly i 5'ATATATATTTTTTTTTTTCTTTTTTTTTTT3 '
poly j 5'ATATATATTTTTTTTTTTTCTTTTTTTTTT3 '
[0047]
Name Fluorescence quenching probe of the present invention
probe a 3'TATATATAAAAAAAACAA5'-BODIPY FL / C6
probe b 3'TATATATAAAAAAAAACA5'-BODIPY FL / C6
probe c 3'TATATATAAAAAAAAAAC5'-BODIPY FL / C6
probe d 3'TATATATAAAAAAAAAAA5'-BODIPY FL / C6
[0048]
Name Fluorescence quenching probe of the present invention
probe f 3'TATATATAAAAAAAAGAA5'-BODIPY FL / C6
probe g 3'TATATATAAAAAAAAAGA5'-BODIPY FL / C6
probe h 3'TATATATAAAAAAAAAAG5'-BODIPY FL / C6
[0049]
(1) Components of the hybridization solution
Synthetic DNA 320 nM (final concentration)
Nucleic acid probe 80nM (final concentration)
NaCl 50 mM (final concentration)
MgCl₂                                       1 mM (final concentration)
Tris-HCl buffer (pH = 7.2) 100 mM (final concentration)
Milli-Q pure water 1.6992ml
Final volume 2.000ml
(2) Hybridization temperature: 51 ° C
(3) Measurement conditions:
Excitation light: 543 nm
Measurement fluorescence color: 569 nm
[0050]
[Table 1]

[0051]
The results are shown in Table 1. As can be seen from Table 1, when a fluorescence quenching probe labeled with a fluorescent dye hybridizes to a target gene, a GC pair is formed at the terminal base pair part where the probe and target gene hybridized at the terminal part. It is preferable that the base sequence of the probe is designed so that at least one base or more of G (guanine) exists in the base sequence of the target gene at a distance of 1 to 3 bases from the terminal base pair portion. It is shown that.
[0052]
Example 2
A target gene (deoxyribooligonucleotide) having the following base sequence and the fluorescence quenching probe of the present invention were prepared. The influence of the number of G in the target gene and the number of G in the nucleic acid probe of the present invention was examined in the same manner as in the above Example, and the results are summarized in Table 2.
[0053]
Name Target gene
poly k 5'TATATATATATTTTTGGGGG3 '
poly l 5'TATATATATATTTTTTGGGG3 '
poly m 5'TATATATATTTTTTTTTGGG3 '
poly n 5'TATATATATTTTTTTTTTGG3 '
poly o 5'TATATATATTTTTTTTTTTG3 '
[0054]
Name Target gene
poly p 5'TATATATATATTTTTCCCCC3 '
poly q 5'TATATATATATTTTTTCCCC3 '
poly r 5'TATATATATTTTTTTTTCCC3 '
poly s 5'TATATATATTTTTTTTTTCC3 '
poly t 5'TATATATATTTTTTTTTTTC3 '
poly u 5'TATATATATTTTTTTTTTTT3 '
[0055]
Name Fluorescence quenching probe
probe k 3'ATATATATATAAAAACCCCC5'-BODIPY FL / C6
probe l 3'ATATATATATAAAAAACCCC5'-BODIPY FL / C6
probe m 3'ATATATATATAAAAAAACCC5'-BODIPY FL / C6
probe n 3'ATATATATATAAAAAAAACC5'-BODIPY FL / C6
probe o 3'ATATATATATAAAAAAAAAC5'-BODIPY FL / C6
[0056]
Name Fluorescence quenching probe
probe p 3'ATATATATATAAAAAGGGGG5'-BODIPY FL / C6
probe q 3'ATATATATATAAAAAAGGGG5'-BODIPY FL / C6
probe r 3'ATATATATATAAAAAAAGGG5'-BODIPY FL / C6
probe s 3'ATATATATATAAAAAAAAGG5'-BODIPY FL / C6
probe t 3'ATATATATATAAAAAAAAAG5'-BODIPY FL / C6
probe u 3'ATATATATATAAAAAAAAAA5'-BODIPY FL / C6
[0057]
[Table 2]

[0058]
As can be seen from Table 2, when the fluorescence quenching probe hybridizes to the target gene, the base sequence of the probe is such that the base pair of the hybridized product forms at least one pair of G and C at the terminal portion. It can be seen that it is suitable to be designed.
[0059]
Example 3
A target gene having the following base sequence and a fluorescence quenching probe were prepared. The influence of the base species in the target gene and the base species in the fluorescence quenching probe was examined in the same manner as in the above Example, and the results are summarized in Table 3.
[0060]
Name Target gene
poly W 5'CCCCCCTTTTTTTTTTTT 3 '
poly X 5'GGGGGGAAAAAAAAAAAA 3 '
poly Y 5'TTTTTTCCCCCCCCCCCC 3 '
poly Z 5'AAAAAAGGGGGGGGGGGG 3 '
[0061]
Name Fluorescence quenching probe
probe w BODIPY FL / C6-5'AAAAAAAAAGGGGGG 3 '
probe x BODIPY FL / C6-5'TTTTTTTTTCCCCCC 3 '
probe y BODIPY FL / C6-5'GGGGGGGGGAAAAAA 3 '
probe z BODIPY FL / C6-5'CCCCCCCCCTTTTTT 3 '
[0062]
[Table 3]

From Table 3, it can be seen that G is deeply involved in the decrease in the fluorescence intensity of the fluorescence quenching probe of the present invention.
[0063]
As a result, from the above example, when (A) the end of the fluorescence quenching probe labeled with a fluorescent dye is composed of C and a target gene is hybridized to form a GC pair, (B) When the end of the fluorescence quenching probe to be labeled is composed of a base other than C, the 3 ′ end side of the target gene from the terminal base pair of the base labeled with the fluorescent dye and the base of the target gene It can be seen that the fluorescence intensity decrease rate is large when at least one G is present in.
[0064]
Example 4
The kind of the dye labeled on the fluorescence quenching probe of the present invention was examined in the same manner as in the above example. In addition, the fluorescence quenching probe z of the said Example 3 was used for the said probe, and the target gene z of the said Example 3 was used for the target gene.
The results are shown in Table 4. As can be seen from the table, suitable fluorescent dyes for use in the present invention include FITC, BODIPY FL, BODIPY FL / C3, 6-joe, TMR and the like.
[0065]
[Table 4]

[0066]
Example 5
Preparation of fluorescence quenching probes Eu47F and Eu1392R modified with fluorescent dye BODIPY FL
(5-1) Synthesis of fluorescence quenching probe Eu47F
(5 ′) The fluorescence quenching probe Eu47F labeled with bodepy FL in the same manner as in Example 1 on the phosphate group at the 5 ′ end of deoxyribooligonucleotide having the base sequence of CITAACACATGCAAGTCG (3 ′) (I = inosine) Synthesized.
[0067]
(5-2) Synthesis of fluorescence quenching probe Eu1392R
Using the deoxyribooligonucleotide having the base sequence of (5 ′) TTGTACACACCGCCCGTCA (3 ′), the fluorescence quenching probe Eu1392R of the present invention was synthesized in the same manner as in the above (5-1).
[0068]
Example 6
(6-1) Cultivation of E. coli strain JM109
E. coli strain JM109 was cultured in 53 medium (composition: casein peptone (casein digested with trypsin), 10 g; yeast extract, 5 g; glucose, 5 g; salt, 5 g; distilled water, 1000 mL) (medium 50 mL / 250 mL conical) Flask, 37 ° C., 12 hours, shaking culture). Then, the cells were collected from the culture solution (centrifugation 10,000 rpm, 5 minutes, washed twice with distilled water).
[0069]
(6-2) Preparation of 16S rRNA cDNA
Total RNA was extracted from the bacterial cells using the SOGEN kit (Nippon Gene) according to the protocol of this kit.
Then BcaBEST^TMUsing the RNA PCR kit (Takara Shuzo Co., Ltd.), according to the protocol of this kit, amplification and reverse transcription reaction (RT-PCR) for 16sRNA were performed on the extract under known normal conditions. At that time, the fluorescence quenching probe Eu1392R of the present invention was used as a primer. Subsequently, RNA was digested with Rnase H (30 ° C., 20 minutes) to obtain a pure cDNA of 16S rRNA gene. cDNA concentration at OliGreen^RMeasured using ssDNA Quantitation kit (Molecular Probes).
[0070]
Example 7
(7-1) Quantitative PCR, data analysis, and creation of cDNA calibration curve
The cDNA solution was subjected to a real-time monitoring quantitative PCR reaction using the fluorescence quenching probes Eu47F and Eu1392R of the present invention as a plumer. The former was a forward type primer, and the latter was a reverse type or reverse type primer.
LightCycler as a real-time monitoring quantitative PCR device^TMUsing Sytem (Roche Diagnostics, Germany), the reaction was carried out according to the procedure described in the procedure manual. As DNA polymeraseTaKaRaTaq ^TM (Takara Shuzo Co., Ltd.) was used.
[0071]
PCR was performed with the following components.

The cDNA was tested with the copy number in the experimental group shown in the note of FIG. MgCl₂The final concentration of was 2 mM.
[0072]
The reaction conditions were as follows.

The measurement conditions were as follows.
Excitation light: 543 nm
Measurement fluorescence color: 569nm
[0073]
Real-time monitoring quantitative PCR was performed under the above conditions to measure the fluorescence intensity of each cycle. The measured values were analyzed according to the data analysis method of the present invention. That is, the data was processed in the following process.
(A) The fluorescence intensity value of the reaction system when the amplified nucleic acid in each cycle was hybridized with a nucleic acid primer labeled with a fluorescent dye (that is, the fluorescence intensity value at the end of the nucleic acid extension reaction (72 ° C.)) was amplified. The correction calculation process divided by the fluorescence intensity value of the reaction system when the nucleic acid hybridized with the nucleic acid primer is completely dissociated (that is, the fluorescence intensity value at the end of the nucleic acid heat denaturation reaction (96 ° C.)), The fluorescence intensity value was corrected by [Equation 1].
[Equation 8]
f_n= F_{hyb, n}/ F_{den, n}      ... [Formula 1]
[Where f_n= Cycle fluorescence intensity correction value, f_{hyb, n}= 72 ° C fluorescence intensity value for each cycle, f_{den, n}= 96 ° C fluorescence intensity value of each cycle]
[0074]
(B) Substituting the correction calculation processing value in [Formula 1] in each cycle into [Formula 3], and calculating the fluorescence quenching rate between samples in each cycle, that is, in the following [Formula 10] The process of computing,
[Equation 9]
F_n= F_n/ F_{twenty five}      ... [Formula 10]
[Where F_n= Arithmetic processing value of each cycle, f_n= Value of each cycle obtained by [Equation 1], f = value obtained by [Equation 1] and the number of cycles is 25th].
[Equation 10] is obtained when a = 25 in [Equation 3].
[0075]
(C) The process of calculating the logarithmic value of the change rate (decrease rate or quenching rate) of the fluorescence intensity according to [Equation 6] from the calculation process value of each cycle obtained in the process of (b), that is, The calculation process of [Formula 11] of
[Expression 10]
log_Ten{(1-F_n) × 100} [Formula 11]
[Where F_n= [Value obtained by [Equation 10]].
[Formula 11] is obtained when b = 10 and A = 100 in [Formula 6].
[0076]
The results are shown in FIG.
FIG. 1 shows the values calculated in the processes (a), (b) and (c) plotted against the number of cycles and printed.
[0077]
Next, it processed in the process of following (d) and (e) based on the graph of FIG.
(D) A process of thresholding 0.2 of the data processed in the process of (c) and calculating the number of cycles reaching the value.
(E) A process of preparing a graph in which the value calculated in the process of (d) is plotted on the X axis and the copy number before starting the reaction is plotted on the Y axis, that is, a calibration curve of E. coli cDNA (FIG. 2).
FIG. 2 shows that data obtained by the quantitative PCR method of the present invention was processed in the data analysis method of the present invention, that is, in the process of (a), (b), (c), (d), and (e). The final result. It can be seen from FIG. 2 that the copy number before starting the reaction can be obtained with high accuracy for the nucleic acid sample having an unknown copy number.
[0078]
Example 8
(8-1) Construction of polymorphic system (complex microbial system)
(Ten types of bacterial strains shown in Table 5 were purchased from DSMZ (Deutshe Sammlung von Mikroorganismen und Zellkulturen GmbH) and cultured separately for each strain using the above-mentioned 53 medium. The bacterial cells were collected from each culture solution (centrifugation 10,000 rpm, 10 minutes, washed twice with distilled water) For each bacterial cell, the SOGEN kit (Nippon Gene) was used in the same manner as described above. To extract total RNA.
[0079]

[0080]
1: Paracoccus pantotrophus
2: Sphingomonas natatoria
3: Bdellovibrio stolpii
4: Microbacterium imperiale
5: Pseudomonas fluorescens
6: Agromyces medislanum
7: Cellulomonas cellulans
8: Brevibacterium liquefaciens
9: Leminorella grimontii
10: Rhodococcus luteus
[0081]
Thereafter, pure cDNA of 16S rRNA gene of each strain was obtained in the same manner as in the case of E. coli. The cDNA concentration of each of the obtained 10 strains was measured in the same manner as in the case of E. coli. The solution whose cDNA concentration was found was diluted with distilled water to 300,000 copies / μL. For 10 strains, a mixture of the diluted solutions in an equivalent amount was defined as a complex microbial system, that is, a polymorphic system (hereinafter referred to as a polymorphic system). In this polymorphism system, 10 strains of cDNA are contained at a concentration of 300,000 copies / μL, respectively, and as a whole, a concentration cDNA of 3,000,000 copies / μL is contained.
[0082]
(8-2) Real-time monitoring quantitative PCR
For the polymorphic cDNA, real-time monitoring quantitative PCR was performed in the same manner as in E. coli using the fluorescence quenching probes Eu47F and Eu1392R of the present invention as a common strainer.
The polymorphic sample was added to the reaction solution in an absolute amount of 300,000 copies / 20 μL (20 μL of the entire reaction solution). In the real-time monitoring quantitative PCR of the polymorphic system, a decrease in fluorescence intensity was observed, and the reaction was stopped at the number of 22 cycles which is an exponential amplification phase of the gene (see FIG. 1). When the threshold was set as log Rn (fluorescence quenching rate) = 0.2, the copy number of cDNA in the reaction solution of the real-time monitoring quantitative PCR performed on the polymorphic system was 288,000 copies (FIG. 2). Since the initial addition amount, that is, the theoretical value is 300,000 copies, it was confirmed that the calibration curve prepared by the method of the present invention showed good quantitativeness.
[0083]
Example 9
Polymorphism analysis
(9-1) Analysis by T-RFLP
After performing the PCR reaction as described above, the amplified product was purified using a column (MicroconPCR, Millipore Corporation Bedford, MA, USA). The purified product was treated with restriction enzyme Hha1 (recognition site: GCG / C, / = cutting site) O / N (overnight). After the treatment, only the cleaved fragment was purified with a column (Microcon and Micropure-EZ, Millipore Corporation Bedford, MA, USA). The size of the cDNA fragment of each strain after the restriction enzyme treatment is shown in Table 5.
The column-purified cDNA solution was subjected to heat denaturation treatment and then subjected to T-RFLP analysis with a sequencer (ABI PRISMTH 310, PE Applied Biosystems). The peak pattern is shown in FIG. Each peak was quantified using a standard BODIPY FL modified fragment of known concentration. As a result of obtaining the molar composition ratio of each peak, all the molar composition ratios were within the range of 9.4 to 10.8, and no extreme difference in PCR amplification efficiency was observed (see Table 5). The total cDNA copy number determined by quantitative PCR was multiplied by the molar composition ratio to determine the initial cDNA copy number of each strain (see Table 5). The copy number / initial addition copy determined by quantification was 0.89 to 1.04 (see Table 5). Therefore, it was found that the initial copy of the polymorphism in the polymorphism system can be accurately quantified by this method.
[0084]
【The invention's effect】
Since the quantitative polymorphism analysis method of the present invention is configured as described above, it has the following effects.
1) The amount of the target gene and the composition ratio of the polymorphism of the gene can be measured simply, rapidly and with good quantitativeness.
2) One of the quantitative PCR methods of the present invention is novel and has excellent quantitative properties.
3) One of the analysis methods for analyzing the data obtained by the quantitative PCR method is also a novel method, and the initial gene copy number before the PCR reaction can be accurately determined.
4) In the novel quantitative PCR method of the present invention, the amplified nucleic acid is labeled with a fluorescent dye. Therefore, in the analysis of polymorphism, the fluorescent dye can be analyzed as a marker.
5) As a result of 2) to 4), the polymorphic analysis method is particularly excellent in quantification.
[Brief description of the drawings]
FIG. 1 Amplification curve of 16S rRNA gene (cDNA) using quantitative PCR method of the present invention
Solid line: E. coli cDNA; Dotted line: Polymorphic cDNA
10²10^Three10^Four10^Five10⁶: Displays the number of copies.
FIG. 2 is a calibration curve of cDNA prepared by the data analysis method of the present invention.
a: 288000 copies
Fig. 3 Analysis pattern of polymorphic T-RFLP of the present invention
bp: Number of bases (base pair)

Claims (3)

The target gene is amplified by a real-time monitoring quantitative PCR method, the initial concentration of the target gene amount is measured, and for the amplified target gene, T-RFLP (terminal restriction fragment length polymorphism), RFLP (restriction fragment length polymorphism), SSCP (single strand conformation) method, or CFLP (cleavase fragment length polymorphism) method to measure the amount of the initial target gene and the polymorphism ratio or the amount of the gene. in line Ujo quantitative polymorphism analysis method,
A quantitative polymorphism analysis method, characterized in that the real-time monitoring quantitative PCR method is reacted using the following fluorescence quenching probe as a primer, and the amount of decrease in the emission of the fluorescent dye is measured.
Fluorescence quenching probe: 6-joe , BODIPY TMR (trademark), BODIPY FL (trademark), BODIPY FL / C3 (trademark), BODIPY FL / C6 (at the 5 'end or at the phosphate group at the 5' end Trademark), Alexa 488 (trademark), Alexa 532 (trademark) When the probe labeled with the fluorescent dye hybridizes to the target gene at the terminal portion, the terminal base pair of the target gene hybridized to the probe When the base sequence of the probe is designed so that at least one base of G (guanine) exists in the base sequence of the target gene 1 to 3 bases away from the portion, and the probe hybridizes to the target gene, fluorescence A fluorescence quenching probe in which the dye reduces the fluorescence intensity. The real-time monitoring quantitative PCR method, quantitative polymorphism analysis method according to claim 1 is intended to stop the amplification reaction of the target gene in exponential amplification phase. The real-time monitoring quantitative PCR method, to carry out the reaction using a fluorescence quenching probe below as primers, quantitative polymorphism analysis according to claim 1 or 2 which measures the amount of decrease in light emission of the fluorescent dye Method.
Fluorescence quenching probe: 6-joe , BODIPY TMR (trademark), BODIPY FL (trademark), BODIPY FL / C3 (trademark), BODIPY FL / C6 (at the 5 'end or at the phosphate group at the 5' end Trademark), Alexa 488 (trademark), Alexa 532 (trademark) labeled with any fluorescent dye, and when the probe is hybridized to the target gene, a hybrid complex of the probe and the target gene at the end When the base sequence of the probe is designed so that at least a G (guanine) and C (cytosine) pair is formed, and the probe is hybridized to the target gene, Reduce fluorescence quenching probe.

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