JPH1094A5 - - Google Patents

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JPH1094A5
JPH1094A5 JP1997052771A JP5277197A JPH1094A5 JP H1094 A5 JPH1094 A5 JP H1094A5 JP 1997052771 A JP1997052771 A JP 1997052771A JP 5277197 A JP5277197 A JP 5277197A JP H1094 A5 JPH1094 A5 JP H1094A5
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stranded dna
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１）２本鎖ＤＮＡ試料を複数の制限酵素により切断して２本鎖ＤＮＡ断片を得る工程と
２）前記２本鎖ＤＮＡ断片の両３’末端の塩基配列の違いを少なくとも２種類のＤＮＡプライマーの３’末端の１塩基〜４塩基の塩基配列により識別して、前記２本鎖ＤＮＡ断片の両３’末端の塩基配列が所定の塩基配列をもつ前記２本鎖ＤＮＡ断片を、前記２種類のＤＮＡプライマーを用いた相補鎖合成反応を用いて、選択的に分離する工程と、
３）前記相補鎖合成反応の生成物の長さを計測する工程とを有し、
前記ＤＮＡプライマーの１塩基〜４塩基の塩基配列は、アデニン、チミン、グアニン、およびシトシンからの全ての組合せに対応して設けられることを特徴とする核酸分析方法。1) a step of cleaving a double-stranded DNA sample with a plurality of restriction enzymes to obtain double-stranded DNA fragments; and 2) a step of identifying differences in the base sequences at both 3'-ends of the double-stranded DNA fragments based on 1-4 base base sequences at the 3'-ends of at least two types of DNA primers, and selectively separating the double-stranded DNA fragments having predetermined base sequences at both 3'-ends of the double-stranded DNA fragments by a complementary strand synthesis reaction using the two types of DNA primers.
3) measuring the length of the product of the complementary strand synthesis reaction,
A method for analyzing nucleic acids, wherein the base sequence of one to four bases of the DNA primer is provided to correspond to all combinations of adenine, thymine, guanine, and cytosine . １）２本鎖ＤＮＡ試料を複数の制限酵素により切断して２本鎖ＤＮＡ断片を得る工程と、
２）前記２本鎖ＤＮＡ断片の両末端にオリゴヌクレオチドを結合する工程と、
３）前記オリゴヌクレオチドの塩基配列、及び前記オリゴヌクレオチドの塩基配列に続く、前記制限酵素が認識する塩基配列の一部又は全部と相補な塩基配列と、３’末端に１塩基〜４塩基からなる選択塩基配列をもつ、標識されたＤＮＡプライマーの複数からなるＤＮＡプライマーセットの複数セットを用いて、異なる前記制限酵素が認識する塩基配列に挟まれた、前記２本鎖ＤＮＡ断片の領域の相補鎖合成反応を行なう工程と、
４）前記相補鎖合成反応の生成物を電気泳動分離する工程とを有し、前記選択塩基配列は、アデニン、チミン、グアニン、およびシトシンからの全ての組合せに対応して設けられることを特徴とする核酸分析方法。1) cleaving a double-stranded DNA sample with a plurality of restriction enzymes to obtain double-stranded DNA fragments;
2) binding oligonucleotides to both ends of the double-stranded DNA fragment;
3) performing a complementary strand synthesis reaction for a region of the double-stranded DNA fragment sandwiched between different base sequences recognized by the restriction enzymes, using a plurality of DNA primer sets each comprising a plurality of labeled DNA primers each having a base sequence of the oligonucleotide, a base sequence complementary to a part or all of the base sequence recognized by the restriction enzyme following the base sequence of the oligonucleotide, and a selected base sequence consisting of 1 to 4 bases at the 3'end;
and 4) a step of electrophoretically separating the products of the complementary strand synthesis reaction, wherein the selected base sequences are provided corresponding to all combinations of adenine, thymine, guanine, and cytosine . １）２本鎖ＤＮＡ試料を第１の制限酵素により切断して２本鎖ＤＮＡ断片を得る工程と、
２）前記２本鎖ＤＮＡ断片の両末端に第１のオリゴヌクレオチドを結合する工程と、
３）工程２）の生成物を第２の制限酵素により切断して２本鎖ＤＮＡ断片を得る工程と、
４）工程３）で得た前記２本鎖ＤＮＡ断片の末端に第２のオリゴヌクレオチドを結合する工程と、
５）前記第１、第２のオリゴヌクレオチドをそれぞれ両末端に有する前記２本鎖ＤＮＡ断片を捕捉する工程と、
６）工程５）で捕捉された前記２本鎖ＤＮＡ断片を１本鎖ＤＮＡ断片にした後、前記１本鎖ＤＮＡ断片を含む液を複数の容器に分画する工程と、
７）前記第１のオリゴヌクレオチドの塩基配列、及び前記第１のオリゴヌクレオチドの塩基配列に続く、前記第１の制限酵素が認識する塩基配列の一部又は全部と相補な塩基配列と、３’末端に１塩基〜４塩基からなる第１の選択塩基配列をもつ、標識された第１のＤＮＡプライマーの複数からなる第１のＤＮＡプライマーセットと、前記第２のオリゴヌクレオチドの塩基配列、及び前記第２のオリゴヌクレオチドの塩基配列に続く、前記第２の制限酵素が認識する塩基配列の一部又は全部と相補な塩基配列と、３’末端に１塩基〜４塩基からなる第２の選択塩基配列をもつ、標識された第２のＤＮＡプライマーの複数からなる第２のＤＮＡプライマーセットとから、前記第１のＤＮＡプライマーと前記第２のＤＮＡプライマーとの組合わせからなるＤＮＡプライマーを、前記各組合わせに対応させて前記各容器に添加して、前記第１、第２の制限酵素が認識する塩基配列に挟まれた、前記２本鎖ＤＮＡ断片の領域の相補鎖合成反応を、前記各容器で行なう工程と、
８）前記各容器での前記相補鎖合成反応の生成物を電気泳動分離する工程とを有し、前記第 １ の選択塩基配列と前記第 ２ の選択塩基配列は、アデニン、チミン、グアニン、およびシトシンからの全ての組合せに対応して各々設けられることを特徴とする核酸分析方法。1) cleaving a double-stranded DNA sample with a first restriction enzyme to obtain double-stranded DNA fragments;
2) binding a first oligonucleotide to both ends of the double-stranded DNA fragment;
3) cleaving the product of step 2) with a second restriction enzyme to obtain double-stranded DNA fragments;
4) binding a second oligonucleotide to the termini of the double-stranded DNA fragment obtained in step 3);
5) capturing the double-stranded DNA fragment having the first and second oligonucleotides at both ends thereof;
6) converting the double-stranded DNA fragments captured in step 5) into single-stranded DNA fragments, and then fractionating a liquid containing the single-stranded DNA fragments into a plurality of containers;
7) adding to each of the containers DNA primers consisting of combinations of the first DNA primer and the second DNA primer from a first DNA primer set consisting of a plurality of labeled first DNA primers, each having the base sequence of the first oligonucleotide, a base sequence complementary to a part or all of the base sequence recognized by the first restriction enzyme following the base sequence of the first oligonucleotide, and a first selected base sequence consisting of 1 to 4 bases at its 3'-end, and a second DNA primer set consisting of a plurality of labeled second DNA primers, each having the base sequence of the second oligonucleotide, a base sequence complementary to a part or all of the base sequence recognized by the second restriction enzyme following the base sequence of the second oligonucleotide, and a second selected base sequence consisting of 1 to 4 bases at its 3'-end, in correspondence with each of the combinations, and performing a complementary strand synthesis reaction in each of the containers for a region of the double-stranded DNA fragment sandwiched between the base sequences recognized by the first and second restriction enzymes;
8) A step of electrophoretically separating the products of the complementary strand synthesis reaction in each of the containers, wherein the first selected base sequence and the second selected base sequence are provided so as to correspond to all combinations of adenine, thymine, guanine, and cytosine . ビーズ固定
１ａ）２本鎖ＤＮＡ試料を第１の制限酵素により切断して２本鎖ＤＮＡ断片を担体に保持する工程と、
２ａ）前記２本鎖ＤＮＡ断片の末端に第１のオリゴヌクレオチドを結合する工程と、
３ａ）工程２ａ）の生成物を第２の制限酵素により切断して２本鎖ＤＮＡ断片を得る工程と、
４ａ）工程３ａ）で得た前記２本鎖ＤＮＡ断片の末端に第２のオリゴヌクレオチドを結合する工程と、
１ｂ）前記２本鎖ＤＮＡ試料を前記第２の制限酵素により切断して２本鎖ＤＮＡ断片を担体に保持する工程と、
２ｂ）工程１ｂ）で得た前記２本鎖ＤＮＡ断片の末端に前記第２のオリゴヌクレオチドを結合する工程と、
３ｂ）工程２ｂ）の生成物を前記第１の制限酵素により切断して２本鎖ＤＮＡ断片を得る工程と、
４ｂ）工程３ｂ）で得た前記２本鎖ＤＮＡ断片の末端に前記第１のオリゴヌクレオチドを結合する工程と、
５）工程４ａ）及び工程４ｂ）の生成物を混合して、前記担体に保持された２本鎖ＤＮＡ断片を除去した後に、前記２本鎖ＤＮＡ断片を１本鎖ＤＮＡ断片にした後、前記１本鎖ＤＮＡ断片を含む液を複数の容器に分画する工程と、
６）前記第１のオリゴヌクレオチドの塩基配列、及び前記第１のオリゴヌクレオチドの塩基配列に続く、前記第１の制限酵素が認識する塩基配列の一部又は全部と相補な塩基配列と、３’末端に１塩基〜４塩基からなる第１の選択塩基配列をもつ、標識された第１のＤＮＡプライマーの複数からなる第１のＤＮＡプライマーセットと、前記第２のオリゴヌクレオチドの塩基配列、及び前記第２のオリゴヌクレオチドの塩基配列に続く、前記第２の制限酵素が認識する塩基配列の一部又は全部と相補な塩基配列と、３’末端に１塩基〜４塩基からなる第２の選択塩基配列をもつ、標識された第２のＤＮＡプライマーの複数からなる第２のＤＮＡプライマーセットとから、前記第１のＤＮＡプライマーと前記第２のＤＮＡプライマーとの組合わせからなるＤＮＡプライマーを、前記各組合わせに対応させて前記各容器に添加して、前記第１、第２の制限酵素が認識する塩基配列に挟まれた、前記２本鎖ＤＮＡ断片の領域の相補鎖合成反応を、前記各容器で行なう工程と、
７）前記各容器での前記相補鎖合成反応の生成物を電気泳動分離する工程とを有し、前記第 １ の選択塩基配列と前記第 ２ の選択塩基配列は、アデニン、チミン、グアニン、およびシトシンからの全ての組合せに対応して各々設けられることを特徴とする核酸分析方法。Bead immobilization 1a) a step of cleaving a double-stranded DNA sample with a first restriction enzyme and retaining double-stranded DNA fragments on a carrier;
2a) binding a first oligonucleotide to the termini of the double-stranded DNA fragment;
3a) cleaving the product of step 2a) with a second restriction enzyme to obtain double-stranded DNA fragments;
4a) ligating a second oligonucleotide to the termini of the double-stranded DNA fragment obtained in step 3a);
1b) cleaving the double-stranded DNA sample with the second restriction enzyme and retaining the double-stranded DNA fragments on a carrier;
2b) binding the second oligonucleotide to the termini of the double-stranded DNA fragment obtained in step 1b);
3b) cleaving the product of step 2b) with the first restriction enzyme to obtain a double-stranded DNA fragment;
4b) binding the first oligonucleotide to the termini of the double-stranded DNA fragment obtained in step 3b);
5) mixing the products of step 4a) and step 4b), removing the double-stranded DNA fragments retained on the carrier, converting the double-stranded DNA fragments into single-stranded DNA fragments, and then fractionating the liquid containing the single-stranded DNA fragments into a plurality of containers;
6) adding to each of the containers DNA primers consisting of combinations of the first DNA primer and the second DNA primer from a first DNA primer set consisting of a plurality of labeled first DNA primers, each having the base sequence of the first oligonucleotide, a base sequence complementary to a part or all of the base sequence recognized by the first restriction enzyme following the base sequence of the first oligonucleotide, and a first selected base sequence consisting of 1 to 4 bases at its 3'-end, and a second DNA primer set consisting of a plurality of labeled second DNA primers, each having the base sequence of the second oligonucleotide, a base sequence complementary to a part or all of the base sequence recognized by the second restriction enzyme following the base sequence of the second oligonucleotide, and a second selected base sequence consisting of 1 to 4 bases at its 3'-end, in correspondence with each of the combinations, and performing a complementary strand synthesis reaction in each of the containers for a region of the double-stranded DNA fragment sandwiched between the base sequences recognized by the first and second restriction enzymes;
7) A step of electrophoretically separating the products of the complementary strand synthesis reaction in each of the containers, wherein the first selected base sequence and the second selected base sequence are provided so as to correspond to all combinations of adenine, thymine, guanine, and cytosine . １）２本鎖ＤＮＡ試料を複数種類の制限酵素により切断して２本鎖ＤＮＡ断片を得る工程と、
２）前記２本鎖ＤＮＡ断片の両末端に複数種類のオリゴヌクレオチドを結合する工程と、
３）前記２本鎖ＤＮＡ断片を含む液を複数の容器に分画する工程と、
４）前記オリゴヌクレオチドの塩基配列、及び前記オリゴヌクレオチドの塩基配列に続く、前記制限酵素が認識する塩基配列の一部又は全部と相補な塩基配列と、３’末端に１塩基〜４塩基からなる選択塩基配列をもつ、標識された第ＤＮＡプライマーの複数からなるＤＮＡプライマーセットの複数セットの、それぞれのセットからの前記ＤＮＡプライマーの組合わせからなるＤＮＡプライマーを、前記各組合わせに対応させて前記各容器に添加して、２種類の前記制限酵素が認識する塩基配列に挟まれた、前記２本鎖ＤＮＡ断片の領域の相補鎖合成反応を、前記各容器で行なう工程と、
５）前記各容器での前記相補鎖合成反応の生成物を電気泳動分離する工程とを有し、前記選択塩基配列は、アデニン、チミン、グアニン、およびシトシンからの全ての組合せに対応して各々設けられることを特徴とする核酸分析方法。1) a step of cleaving a double-stranded DNA sample with multiple types of restriction enzymes to obtain double-stranded DNA fragments;
2) binding multiple types of oligonucleotides to both ends of the double-stranded DNA fragment;
3) fractionating the liquid containing the double-stranded DNA fragments into a plurality of containers;
4) adding to each of the containers DNA primers consisting of combinations of DNA primers from each of a plurality of DNA primer sets each consisting of a plurality of labeled DNA primers having the base sequence of the oligonucleotide and a base sequence complementary to part or all of the base sequence recognized by the restriction enzyme following the base sequence of the oligonucleotide, and a selected base sequence consisting of 1 to 4 bases at the 3'-end, in correspondence with each of the combinations, and performing a complementary strand synthesis reaction in each of the containers for a region of the double-stranded DNA fragment sandwiched between the base sequences recognized by the two types of restriction enzymes;
and 5) a step of electrophoretically separating the products of the complementary strand synthesis reaction in each of the containers, wherein the selected base sequences are provided corresponding to all combinations of adenine, thymine, guanine, and cytosine . 請求項２、３、４、５のいずれかに記載の核酸分析方法に用いるＤＮＡプライマーセットであり、ＤＮＡプライマーセットは複数のＤＮＡプライマーからなり、各ＤＮＡプライマーは、５’Ｎ１Ｎ２…ＮｎＸ１Ｘ２…ＸｍＺ１Ｚ２…ＺｈＹ１Ｙ２…Ｙｋ３’の構造を有し、Ｎ１Ｎ２…Ｎｎ（５≦ｎ≦２７）は、２本鎖ＤＮＡ断片の末端に結合したオリゴヌクレオチドと実質相補な塩基配列であり、Ｎ１Ｎ２…Ｎｎを構成する何れかのヌクレオチドが標識されており、Ｘ１Ｘ２…Ｘｍ（１≦ｍ≦６）は、制限酵素が認識する塩基配列の一部又は全部と相補な塩基配列であり、Ｚ１Ｚ２…Ｚｈ（０≦ｈ≦３）は、複数種類のヌクレオチドとハイブリダイズし得るヌクレオチドアナログであり、Ｙ１Ｙ２…Ｙｋ（１≦ｋ≦４）は、Ａ、Ｃ、Ｇ及びＴの組合わせの（４のｋ乗）の種類からなることを特徴とするＤＮＡプライマーセット。A DNA primer set for use in the nucleic acid analysis method according to any one of claims 2, 3, 4 and 5, wherein the DNA primer set comprises a plurality of DNA primers, each of which has the structure 5'N1N2...NnX1X2...XmZ1Z2...ZhY1Y2...Yk3', wherein N1N2...Nn (5≦n≦27) is a base sequence substantially complementary to an oligonucleotide attached to the end of a double-stranded DNA fragment, and any of the nucleotides constituting N1N2...Nn is labeled, X1X2...Xm (1≦m≦6) is a base sequence complementary to part or all of a base sequence recognized by a restriction enzyme, Z1Z2...Zh (0≦h≦3) is a nucleotide analog capable of hybridizing with a plurality of types of nucleotides, and Y1Y2...Yk (1≦k≦4) is a DNA primer set consisting of (4 to the k power) combinations of A, C, G and T.
JP05277197A 1996-04-16 1997-03-07 Nucleic acid analysis method Expired - Lifetime JP3783315B2 (en)

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