Method for analysis of nucleic acid and DNA primer sets for use therein
First Claim
1. A method for analysis of a nucleic acid which comprises:
- (1) digesting a double-stranded DNA sample with a first restriction enzyme and a second restriction enzyme to obtain double-stranded DNA fragments;
(2) discriminating a difference in base sequences of 1 to 4 bases adjacent to restriction enzyme recognition sites of the double-stranded DNA fragments with first labeled DNA primers and second labeled DNA primers, said first labeled DNA primers and second labeled DNA primers each having a 3'"'"'- and a 5'"'"'-end; and
extending said first labeled DNA primers and said second labeled DNA primers, by a complementary strand extension synthesis reaction, to classify the double-stranded DNA fragments into 16 to 65536 groups,wherein said first labeled DNA primers have all possible combinations of 1 to 4 bases selected from A, C, G and T at the 3'"'"'-end thereof, and said second labeled DNA primers have all possible combinations of 1 to 4 bases selected from A, C, G and T at the 3'"'"'-end thereof;
(3) measuring the lengths of the products produced during said complementary strand extension synthesis reaction, by electrophoresis; and
,(4) classifying measured lengths of the products produced during said complementary strand extension synthesis reaction into 16 to 65536 groups by the differences in base sequences of 1 to 4 bases adjacent to the restriction enzyme recognition sites of the double-stranded DNA fragments to obtain fingerprinting patterns of said DNA fragments, wherein the restriction enzyme recognition sites are recognized by said first and second restriction enzymes, respectively.
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Abstract
The present invention comprises a method for analysis of a nucleic acid which comprises:
(1) a step of digesting a double-stranded DNA sample with a plurality of restriction enzymes to obtain double-stranded DNA fragments;
(2) a step of ligating a plurality of oligonucleotides to the double-stranded DNA fragments respectively at the both ends thereof;
(3) a step of dispensing a solution containing the double-stranded DNA fragments into a plurality of tubes;
(4) a step of adding DNA primers comprising combinations of DNA primers selected from each set of a plurality of DNA primer sets comprising a plurality of labeled primers having a base sequence complementary to the base sequence of oligonucleotide and a part or all of the base sequence contiguous to the base sequence complementary to the base sequence of the oligonucleotide and recognized by the restriction enzymes and a selective base sequence of 1 to 4 bases at the 3'"'"'-end thereof, to the respective tubes corresponding to the combinations and performing a complementary strand synthesis reaction of the region of the double-stranded DNA fragments between the base sequences recognized by the two restriction enzymes; and,
(5) a step of subjecting the products obtained by the complementary strand synthesis reaction to electrophoresis to produce a large number of DNA fragments from the long double stranded DNA sample digested with restriction enzymes and obtain fingerprinting patterns therefrom which enables to inspect the long double-stranded DNA sample.
9 Citations
21 Claims
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1. A method for analysis of a nucleic acid which comprises:
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(1) digesting a double-stranded DNA sample with a first restriction enzyme and a second restriction enzyme to obtain double-stranded DNA fragments; (2) discriminating a difference in base sequences of 1 to 4 bases adjacent to restriction enzyme recognition sites of the double-stranded DNA fragments with first labeled DNA primers and second labeled DNA primers, said first labeled DNA primers and second labeled DNA primers each having a 3'"'"'- and a 5'"'"'-end; and
extending said first labeled DNA primers and said second labeled DNA primers, by a complementary strand extension synthesis reaction, to classify the double-stranded DNA fragments into 16 to 65536 groups,wherein said first labeled DNA primers have all possible combinations of 1 to 4 bases selected from A, C, G and T at the 3'"'"'-end thereof, and said second labeled DNA primers have all possible combinations of 1 to 4 bases selected from A, C, G and T at the 3'"'"'-end thereof; (3) measuring the lengths of the products produced during said complementary strand extension synthesis reaction, by electrophoresis; and
,(4) classifying measured lengths of the products produced during said complementary strand extension synthesis reaction into 16 to 65536 groups by the differences in base sequences of 1 to 4 bases adjacent to the restriction enzyme recognition sites of the double-stranded DNA fragments to obtain fingerprinting patterns of said DNA fragments, wherein the restriction enzyme recognition sites are recognized by said first and second restriction enzymes, respectively.
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2. A method for analysis of nucleic acid which comprises:
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(1) digesting a double-stranded DNA sample with a first restriction enzyme to obtain double-stranded DNA fragments; (2) ligating a first oligonucleotide having known base sequence to both ends of each of said double-stranded DNA fragments; (3) digesting products obtained in step (2) with a second restriction enzyme to obtain double-stranded DNA fragments; (4) ligating a second oligonucleotide having known base sequence to one end or both ends of each of said double-stranded DNA fragments obtained in step (3), to obtain ligated double-stranded DNA fragments; (5) capturing one end of each of the ligated double-stranded DNA fragments each having, at said one end, said first oligonucleotide, and having at another end, said second oligonucleotide; (6) converting the ligated double-stranded DNA fragments captured in step (5) into single-stranded DNA fragments and then dispensing a solution containing the single-stranded DNA fragments into a plurality of tubes; (7) adding a first DNA primer selected from a first labeled DNA primer set and a second primer selected from a second DNA primer set to said respective tubes for each of combinations of said first DNA primer and said second DNA primer, and extending said first DNA primer and said second DNA primer, by a complementary strand extension synthesis reaction in said respective tubes, wherein each of the primers of said first labeled DNA primer set has a 3'"'"'- and a 5'"'"'-end, and has at the 5'"'"'-end of each of the primers of said first labeled DNA primer set, a first base sequence complementary to at least a part of the base sequence recognized by said first restriction enzyme and has, at the 3'"'"'-end, a first selective base sequence of 1 to 4 bases, said first selective base sequence discriminates a difference in base sequences of 1 to 4 bases adjacent to the 5'"'"'-end of the base sequence recognized by said first restriction enzyme in each of the single-stranded DNA fragments, and the primers of said first labeled DNA primer set have all possible combinations of 1 to 4 bases selected from A, C, G and T at the 3'"'"'-end thereof, and wherein each of the primers of said second DNA primer set has a 3'"'"'- and a 5'"'"'-end, and at the 5'"'"'-end, a second base sequence complementary to at least a part of the base sequence recognized by said second restriction enzyme and has, at the 3'"'"'-end, a second selective base sequence of 1 to 4 bases, said second selective base sequence discriminates a difference in base sequences of 1 to 4 bases directly connected to the 5'"'"'-end of the base sequence recognized by said second restriction enzyme in each of the single-stranded DNA fragments, and the primers of second DNA primer set have, at the 3'"'"'-end, all possible combinations of 1 to 4 bases selected from A, C, G and T; (8) measuring the lengths of the products produced during said complementary strand extension synthesis reaction in said respective tubes in step (7), by electrophoresis; and (9) classifying measured lengths of the products produced during said complementary strand extension synthesis reaction into 16 to 65536 groups by the differences in base sequences of 1 to 4 bases adjacent to the restriction enzyme recognition sites of the ligated double-stranded DNA fragments to obtain fingerprinting patterns of said DNA fragments, wherein the restriction enzyme recognition sites are recognized by said first and second restriction enzymes, respectively. - View Dependent Claims (3)
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4. A method for analysis of nucleic acid which comprises:
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(1) digesting a double-stranded DNA sample with a first restriction enzyme and a second restriction enzyme to obtain double-stranded DNA fragments; (2) ligating a single-stranded oligonucleotide having known base sequence to 3'"'"'-ends of each of said double-stranded DNA fragments; (3) dispensing double-stranded DNA fragments obtained in step (2) into a plurality of tubes and then converting said double-stranded DNA fragments obtained in step (2) into single-stranded DNA fragments in said respective tubes to obtain first single-stranded DNA fragments and second single-stranded DNA fragments; (4) adding a first DNA primer selected from a first labeled DNA primer set and a second DNA primer selected from a second labeled DNA primer set to said respective tubes for each of combinations of said first DNA primer and said second DNA primer, and extending said first DNA primer and said second DNA primer hybridized with the single-stranded DNA fragment, by a complementary strand extension synthesis reaction in said respective tubes, wherein each of the primers of said first labeled DNA primer set has a 3'"'"'- and a 5'"'"'-end, and has at the 5'"'"'-end, a first base sequence complementary to at least a part of the base sequence recognized by said first restriction enzyme and has, at the 3'"'"'-end, a first selective base sequence of 1 to 4 bases, said first selective base sequence discriminates a difference in base sequences of 1 to 4 bases adjacent to the 5'"'"'-end of the base sequence recognized by said first restriction enzyme in each of the first single-stranded DNA fragments, and the primers of said first labeled DNA primer set have, at the 3'"'"'-end, all possible combinations of 1 to 4 bases selected from A, C, G and T, and wherein each of the primers of said second labeled DNA primer set has a 3'"'"'- and a 5'"'"'-end, and has at the 5'"'"'-end, a second base sequence complementary to at least a part of the base sequence recognized by said second restriction enzyme and has, at the 3'"'"'-end, a second selective base sequence of 1 to 4 bases, said second selective base sequence discriminates a difference in base sequences of 1 to 4 bases adjacent to the 5'"'"'-end of the base sequence recognized by said second restriction enzyme in the second single-stranded DNA fragments, and the primers of said second labeled DNA primer set have, at the 3'"'"'-end, all possible combinations of 1 to 4 bases selected from A, C, G and T; (5) measuring the lengths of products produced during said complementary strand extension synthesis reaction in said respective tubes in step (4), by electrophoresis; and
,(6) classifying measured lengths of the products produced during said complementary strand extension synthesis reaction into 16 to 65536 groups by the differences in base sequences of 1 to 4 bases adjacent to the restriction enzyme recognition sites of the double-stranded DNA fragments obtained in step (2) to obtain fingerprinting patterns of said DNA fragments, wherein the restriction enzyme recognition sites are recognized by said first and second restriction enzymes, respectively. - View Dependent Claims (5, 6, 7, 8, 9)
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10. A method for analysis of nucleic acid which comprises:
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(1) digesting a double-stranded DNA sample with a first restriction enzyme and a second restriction enzyme to obtain double-stranded DNA fragments, (2) discriminating a difference in base sequences of 1 to 4 bases adjacent to restriction enzyme recognition sites of the double-stranded DNA fragments with first labeled DNA primers and second DNA primers, and extending said first labeled DNA primers and said second DNA primers, by a complementary strand extension synthesis reaction, to classify the double-stranded DNA fragments into 16 to 65536 groups, wherein said first labeled DNA primers have a 3'"'"'- and a 5'"'"'-end; and
have at the 3'"'"'-end, all possible combinations of 1 to 4 bases selected from A, C, G and T, and said second DNA primers have, at the 3'"'"'-end, all possible combinations of 1 to 4 bases selected from the A, C, G and T;(3) measuring the lengths of products produced during said complementary strand extension synthesis reaction, by electrophoresis; and (4) classifying measured lengths of the products produced during said complementary strand extension synthesis reaction into 16 to 65536 groups by the differences in base sequences of 1 to 4 bases adjacent to the restriction enzyme recognition sites of the double-stranded DNA fragments to obtain fingerprinting patterns of said DNA fragments, wherein the restriction enzyme recognition sites are recognized by said first and second restriction enzymes.
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11. A method for analysis of nucleic acid which comprises:
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(1) digesting a double-stranded DNA sample with a first restriction enzyme and a second restriction enzyme to obtain double-stranded DNA fragments; (2) discriminating a difference in base sequences of 1 to 4 bases adjacent to restriction enzyme recognition sites of the double-stranded DNA fragments with first labeled DNA primers and second labeled DNA primers, and extending, by a complementary strand extension synthesis reaction, each of first labeled DNA primers hybridized with each of first single-stranded DNA fragments obtained from the double-stranded DNA fragments and each of second labeled DNA primers hybridized with each of second single-stranded DNA fragments obtained from the double-stranded DNA fragments, to classify the double-stranded DNA fragments into 16 to 65536 groups, wherein said first labeled DNA primers have a 3'"'"'- and a 5'"'"'-end; and
have at the 3'"'"'-end, all possible combinations of 1 to 4 bases selected from A, C, G and T, and said second labeled DNA primers each have, at the 3'"'"'-end, all possible combinations of 1 to 4 bases selected from A, C, G and T;(3) measuring the lengths of products produced during said complementary strand extension synthesis reaction, by electrophoresis; and
,(4) classifying measured lengths of the products produced during said complementary strand extension synthesis reaction into 16 to 65536 groups by the differences in bases sequences of 1 to 4 bases adjacent to the restriction enzyme recognition sites of the double-stranded DNA fragments to obtain fingerprinting patterns of said DNA fragments, wherein the restriction enzyme recognition sites are recognized by said first and second restriction enzymes.
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12. A method for analysis of nucleic acid which comprises:
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(1) dispensing double-stranded DNA samples into a first fraction and a second fraction; (2) digesting said double-stranded DNA samples of said first fraction with a first restriction enzyme to obtain double-stranded DNA fragments and capturing one end of said double-stranded DNA fragments on a first support; (3) ligating a first oligonucleotide having a known base sequence to another end of each of said double-stranded DNA fragments captured on said first support; (4) digesting said double-stranded DNA fragments captured and ligated in step (3) with a second restriction enzyme to obtain double-stranded DNA fragments; (5) removing said first support from said first fraction to remove the double-stranded DNA fragments captured on said first support; (6) ligating a second oligonucleotide having a known base sequence to one end or both ends of each of said double-stranded DNA fragments remaining in the first fraction after steps (4) and (5), to obtain first double-stranded DNA fragments; (7) digesting said double-stranded DNA samples of said second fraction with said second restriction enzyme and capturing one end of the double-stranded DNA fragments on a second support; (8) ligating said second oligonucleotide to another end of each of said double-stranded DNA fragments captured on said second support; (9) digesting said double-stranded DNA fragments captured and ligated in step (8) with said first restriction enzyme to obtain double-stranded DNA fragments; (10) removing said second support from said second fraction to remove the double-stranded DNA fragments captured on said second support; (11) ligating said first oligonucleotide to one end or both ends of said double-stranded DNA fragments remaining in the second fraction after steps (9) and (10), to obtain second double-stranded DNA fragments; (12) dispensing a mixture of said first double-stranded DNA fragments obtained in step (6) and said second double-stranded DNA fragments obtained in step (11) into a plurality of tubes, and then converting said first and second double-stranded DNA fragments into single-stranded DNA fragments in said respective tubes to obtain first single-stranded DNA fragments and second single-stranded DNA fragments; (13) adding a first DNA primer selected from a first labeled DNA primer set and a second DNA primer selected from a second labeled DNA primer set to said respective tubes for each of combinations of said first DNA primer and said second DNA primer, and extending said first DNA primer hybridized with the first single-stranded DNA fragment and said second DNA primer hybridized with the second single-stranded DNA fragment, by a complementary strand extension synthesis reaction in said respective tubes, wherein each of primers of said first labeled DNA primer set has a 3'"'"'- and a 5'"'"'-end, and has at the 5'"'"'-end, a first base sequence complementary to at least a part of the base sequence recognized by said first restriction enzyme and has, at the 3'"'"'-end, a first selective base sequence of 1 to 4 bases, said first selective base sequence discriminates a difference in base sequences of 1 to 4 bases adjacent to the 5'"'"'-end of the base sequence recognized by said first restriction enzyme in each of the first single-stranded DNA fragments, and the primers of said first labeled DNA primer set have, at the 3'"'"'-end, all possible combinations of 1 to 4 bases selected from A, C, G and T, and wherein each of the primers of said second labeled DNA primer set has a 3'"'"'- and a 5'"'"'-end, and has at the 5'"'"'-end, a second base sequence complementary to at least a part of the base sequence recognized by said second restriction enzyme and has, at the 3'"'"'-end, a second selective base sequence of 1 to 4 bases, said second selective base sequence discriminates a difference in base sequences of 1 to 4 bases adjacent to the 5'"'"'-end of the base sequence recognized by said second restriction enzyme in the second single-stranded DNA fragments, and the primers of said second labeled DNA primer set have, at the 3'"'"'-end, all possible combinations of 1 to 4 bases selected from A, C, G and T; (14) measuring the lengths of products produced during said complementary strand extension synthesis reaction, by electrophoresis; and
,(15) classifying measured lengths of the products produced during said complementary strand extension synthesis reaction into 16 to 65536 groups by the differences in base sequences of 1 to 4 bases adjacent to the restriction enzyme recognition sites of the first double-stranded DNA fragments and the second double-stranded DNA fragments to obtain fingerprinting patterns of said DNA fragments, wherein the restriction enzyme recognition sites are recognized by said first and second restriction enzymes. - View Dependent Claims (13, 14, 15, 16)
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17. A method for analysis of nucleic acid which comprises:
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(1) digesting a double-stranded DNA sample with a plurality of restriction enzymes to obtain double-stranded DNA fragments; (2) ligating a single-stranded oligonucleotide having known base sequence to 3'"'"'-ends of each of said double-stranded DNA fragments; (3) dispensing double-stranded DNA fragments obtained in step (2) into a plurality of tubes and then converting said double-stranded DNA fragments obtained in step (2) into single-stranded DNA fragments in said respective tubes to obtain first single-stranded DNA fragments and second single-stranded DNA fragments; (4) adding a first DNA primer selected from a first DNA primer set which is one set in two sets selected from labeled DNA primer sets and a second DNA primer selected from a second DNA primer set which is another set of the two sets selected from said labeled DNA primer sets, to said respective tubes for each of combinations of the first DNA primer and the second DNA primer, and extending said first DNA primer hybridized with the first single-stranded DNA fragment and said second DNA primer hybridized with the second single-stranded DNA fragment, by a complementary strand extension synthesis reaction in said respective tubes, wherein labeled DNA primer sets correspond to said restriction enzymes, respectively, and the number of labeled DNA primer sets is equal to the number of species of said restriction enzymes, and wherein each of the primers of said first DNA primer set has a 3'"'"'- and a 5'"'"'-end, and has at the 5'"'"'-end, a first base sequence complementary to at least a part of the base sequence recognized by a first restriction enzyme and has, at the 3'"'"'-end, a first selective base sequence of 1 to 4 bases, said first selective base sequence discriminates a difference in base sequences of 1 to 4 bases adjacent to the 5'"'"'-end of the base sequence recognized by said first restriction enzyme in each of the first single-stranded DNA fragments, and the primers of said first DNA primer set have, at the 3'"'"'-end, all possible combinations of 1 to 4 bases selected from A, C, G and T, and wherein each of the primers of said second DNA primer set has a 3'"'"'- and a 5'"'"'-end, and has at the 5'"'"'-end, a second base sequence complementary to at least a part of the base sequence recognized by a second restriction enzyme and has, at the 3'"'"'-end, a second selective base sequence of 1 to 4 bases, said second selective base sequence discriminates a difference in base sequences of 1 to 4 bases adjacent to the 5'"'"'-end of the base sequence recognized by said second restriction enzyme in each of the second single-stranded DNA fragments, and the primers of said second DNA primer set have, at the 3'"'"'-end, all possible combinations of 1 to 4 bases selected from A, C, G and T, (5) measuring the lengths of products produced during said complementary strand extension synthesis reaction in step (4) for all possible combinations of two sets in said labeled DNA primer sets, by electrophoresis; and (6) classifying measured lengths of the products produced during said complementary strand extension synthesis reaction in step (5) into 16 to 65536 groups by the differences in base sequences of 1 to 4 bases adjacent to the restriction enzyme recognition sites of the double-stranded DNA fragments obtained in step (2) to obtain fingerprinting patterns of said DNA fragments, wherein the restriction enzyme recognition sites are recognized by two different restriction enzymes. - View Dependent Claims (18, 19, 20, 21)
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Specification