Method of analysis or assay for polynucleotides and analyzer or instrument for polynucleotides
First Claim
1. A method of analysis or assay for nucleotides which comprises:
- (1) digesting DNA'"'"'s with a restriction enzyme to obtain DNA fragments;
(2) discriminating differences in sequences of two bases adjacent to restriction enzyme recognition sites of the DNA fragments with labeled DNA probes and extending the labeled DNA probes by a complementary strand synthesis to fractionate the DNA fragments into sixteen groups, wherein the labeled DNA probes have all possible sixteen combinations of two bases out of A, C, G and T at the 3'"'"' termini thereof; and
(3) measuring lengths of the DNA fragments which belong to each of said sixteen groups, or lengths of the labeled DNA probes extended by said complementary strand synthesis;
wherein measured lengths of the DNA fragments fractionated into the sixteen groups by the differences in sequences of two bases adjacent to the restriction enzyme recognition sites of the DNA fragments are employed as fingerprints.
1 Assignment
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Accused Products
Abstract
A method of analysis or assay for nucleotides comprises: (1) a step of digesting DNA with a restriction enzyme; (2) a step of discriminating a difference in sequences of the DNA fragments obtained in step (1) above around the 3'"'"' termini thereof with a DNA probe and extending the DNA probe by a complementary strand synthesis to fractionate the DNA fragments into groups; and, (3) a step of measuring lengths of the DNA fragments which belong to said groups, or length of the DNA probe extended by said complementary strand extension reaction; wherein the thus measured lengths obtained for every sequence of the bases of the DNA fragments around the 3'"'"' termini thereof are employed as fingerprints.
10 Citations
28 Claims
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1. A method of analysis or assay for nucleotides which comprises:
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(1) digesting DNA'"'"'s with a restriction enzyme to obtain DNA fragments; (2) discriminating differences in sequences of two bases adjacent to restriction enzyme recognition sites of the DNA fragments with labeled DNA probes and extending the labeled DNA probes by a complementary strand synthesis to fractionate the DNA fragments into sixteen groups, wherein the labeled DNA probes have all possible sixteen combinations of two bases out of A, C, G and T at the 3'"'"' termini thereof; and (3) measuring lengths of the DNA fragments which belong to each of said sixteen groups, or lengths of the labeled DNA probes extended by said complementary strand synthesis; wherein measured lengths of the DNA fragments fractionated into the sixteen groups by the differences in sequences of two bases adjacent to the restriction enzyme recognition sites of the DNA fragments are employed as fingerprints. - View Dependent Claims (2, 4, 5, 7, 8, 9, 10, 14, 15, 16)
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3. A method of analysis or assay for nucleotides which comprises:
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(1) digesting DNA with a restriction enzyme to obtain DNA fragments; (2) introducing an oligomer comprising deoxynucleotides or an oligomer containing analogues of the deoxynucleotides into the DNA fragments at the 3'"'"' termini thereof, by using ligase or terminal deoxynucleotidyl transferase; (3) performing a complementary strand synthesis using a first probe set comprising four labeled DNA probes or a second probe set comprising sixteen labeled DNA probes and complementary strand to the introduced oligomer or to a part of the recognition sequence recognized by the restriction enzyme thereby to extend said labeled DNA probes, wherein one base at 3'"'"' termini of the four labeled DNA probes of the first probe set is composed of all possible four combinations of one base species out of A, C, G and T or analogues thereof, and two bases at the 3'"'"' termini of the sixteen labeled DNA probes of the second probe set are composed of all possible sixteen combinations of two bases species out of A, C, G and T or analogues thereof; and (4) subjecting the strand extended by the complementary strand synthesis to electrophoresis to detect the DNA fragments and to obtain each of four electropherograms or each of sixteen electropherograms; wherein the four electropherograms are classified by differences in sequences of one base adjacent to restriction enzyme recognition sites of the DNA fragments, and the four electropherograms are employed as fingerprints, or the sixteen electropherograms are classified by differences in sequences of two bases adjacent to the restriction enzyme recognition sites of the DNA fragments, and the sixteen electropherograms are employed as fingerprints.
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6. A method of analysis or assay for nucleotides which comprises:
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(1) digesting DNA'"'"'s with a restriction enzyme to obtain DNA fragments; (2) introducing a first and a second oligomer comprising deoxynucleotides or a first and a second oligomer containing analogues of the deoxynucleotides into the DNA fragments at the 3'"'"' and 5'"'"' termini thereof, respectively, by using ligase; (3) performing a complementary strand synthesis using first labeled DNA probes having all possible combinations of 1 to 3 bases out of A, C, G and T or analogues thereof at the 31 termini thereof, the first labeled DNA probes being complementary to the introduced first oligomer or to a part of the recognition sequence recognized by said restriction enzyme, or using labeled nucleotide phosphate, thereby extending the complementary strand of each of said first labeled DNA probes; (4) classifying and recovering the DNA strand extended by the complementary strand synthesis for each of the first labeled DNA probes; (5) performing a complementary strand synthesis using second labeled DNA probes having all possible combinations of 1 to 3 bases out of A, C, G, and T or analogues thereof at the 3'"'"' termini thereof, the second labeled DNA probes being complementary to the DNA strand extended by the complementary strand synthesis in step (3) or to a part of the recognition sequence recognized by said restriction enzyme, or using labeled nucleotide phosphate, thereby extending the complementary strand of each of the second labeled DNA probes; (6) fractionating and detecting the DNA strands extended by the complementary strand synthesis in step (5), depending upon length, for each of the second labeled DNA probes.
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11. A method of analysis or assay for nucleotides which comprises:
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(1) fractionating DNA fragments into sixteen groups by differences in sequences of two bases adjacent to a restriction enzyme recognition site of each of the DNA fragments obtained by digesting a sample DNA with a restriction enzvme, using labeled DNA probes having all possible sixteen combinations of two bases out of A, C, G and T at the 31 termini thereof; and (2) separating the DNA fragments contained in each of the sixteen groups or complementary strands to the DNA fragments by electrophoresis in terms of length; wherein an electropherogram obtained by the electrophoresis is used for analysis or assay. - View Dependent Claims (12)
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13. A method of analysis or assay for DNA which comprises:
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(1) digesting DNA'"'"'s with a restriction enzyme to obtain DNA fragments; (2) discriminating differences in sequences of 1 to 3 bases adjacent to the restriction enzyme recognition sites of the DNA fragments with labeled DNA probes and extending the labeled DNA probes by a complementary strand synthesis to fractionate the DNA fragments into groups, wherein the labeled DNA probes have all possible combinations of 1 to 3 bases out of A, C, G and T at the 3'"'"' termini thereof; and (3) measuring lengths of the DNA fragments which belong to said groups, or lengths of the labeled DNA probes extended by said complementary strand synthesis; wherein measured lengths of the DNA fragments fractionated into the groups by the differences in sequences of 1 to 3 bases adjacent to the restriction enzyme recognition sites of the DNA fragments termini are employed as fingerprints.
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17. A method of analysis or assay for nucleotides which comprises:
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(1) collecting mRNA from a tissue sample to prepare a mRNA-cDNA complementary pair; (2) preparing a double stranded DNA from the mRNA-cDNA complementary pair; (3) digesting the double stranded DNA with a restriction enzyme to obtain DNA fragments; (4) discriminating differences in sequences of two bases adjacent to the restriction enzyme recognition sites of the DNA fragments with labeled DNA probes and extending the labeled DNA probes by a complementary strand synthesis to fractionate the DNA fragments into sixteen groups, wherein the labeled DNA probes have all Possible sixteen combinations of two bases out of A, C, G and T at the 3'"'"' termini thereof; and (5) measuring lengths of the DNA fragments which belong to each of said sixteen groups, or lengths of the labeled DNA probes extended by said complementary strand synthesis; wherein measured lengths of the DNA fragments fractionated into the sixteen groups by the differences in sequence of two bases adjacent to the restriction enzyme recognition sites of the DNA fragments are employed as fingerprints to give information on the kind and amount of said mRNA.
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18. A method of analysis or assay for nucleotides which comprises:
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(1) collecting mRNA from a tissue sample to prepare a mRNA-cDNA complementary pair; (2) preparing a double stranded DNA from the mRNA-cDNA complementary pair; (3) digesting the double stranded DNA with a restriction enzyme to obtain DNA fragments; (4) introducing an oligomer comprising deoxynucleotides or an oligomer containing analogues of the deoxynucleotides into the DNA fragments at the 3'"'"' termini thereof; (5) performing a complementary strand synthesis using a first probe set comprising four labeled DNA probes or a second probe set comprising sixteen labeled DNA probes complementary to the introduced oligomer or to a part of the recognition sequence recognized by the restriction enzyme thereby extending said labeled DNA probes, wherein one base at the 3'"'"' termini of the four labeled DNA probes of the first probe set is composed of all possible four combinations of one base species out of A, C, G and T or analogues thereof, and two bases at the 3'"'"' termini of the sixteen labeled DNA probes of the second probe set are composed of all possible sixteen combinations of two bases species out of A, C, G and T or analogues thereof; and (6) subjecting the strand extended by the complementary strand synthesis to electrophoresis to detect the DNA fragments;
thereby giving information on the kind and amount of said mRNA.
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19. A method of analysis or assay for nucleotides which comprises:
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(1) collecting mRNA from a tissue sample to prepare a mRNA-cDNA complementary pair; (2) preparing a double stranded DNA from the mRNA-cDNA complementary pair; (3) digesting the double stranded DNA with a restriction enzyme to obtain DNA fragments; (4) introducing a first and a second oligomer comprising deoxynucleotides or a first and a second oligomer containing analogues of the deoxynucleotides into the DNA fragments at the 3'"'"' and 5'"'"' termini thereof, respectively, by using ligase; (5) performing a complementary strand synthesis using a first probe set comprising four labeled DNA probes or a second probe set comprising sixteen labeled DNA probes complementary to the introduced oligomer in step (4) or to a part of the recognition sequence recognized by the restriction enzyme, thereby extending said labeled DNA probes of the first or second probe set, wherein one base at the 3'"'"' termini of the four labeled DNA probes of the first probe set is composed of all possible four combinations of one base species out of A, C, G and T or analogues thereof, and two bases at the 3'"'"' termini of the sixteen labeled DNA probes of the second probe set are composed of all possible sixteen combinations of two bases species out of A, C, G and T or analogues thereof; (6) classifying and recovering the DNA strand extended by the complementary strand synthesis, for each of the labeled DNA probes of the first or second probe set; (7) performing a complementary strand synthesis using a third probe set comprising four labeled DNA probes or a fourth probe set comprising sixteen labeled DNA probes complementary to the DNA strand extended by the complementary strand synthesis in step (5) or to a part of the recognition sequence recognized by the restriction enzyme, thereby extending said labeled DNA Probes of the third or fourth probe set, wherein one base at the 3'"'"' termini of the four labeled DNA probes of the third probe set is composed of all possible four combinations of one base species out of A, C, G and T or analogues thereof, and two bases at the 3'"'"' termini of the sixteen labeled DNA probes of the fourth probe set are composed of all possible sixteen combinations of two bases species out of A, C, G and T or analogues thereof, and the third probe set is different from the first probe set and the fourth probe set is different from the second probe set; and (8) fractionating and detecting the DNA strands extended by the complementary strand synthesis in step (7), depending upon length; thereby giving information on the kind and amount of said mRNA.
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20. A method of analysis or assay for nucleotides which comprises:
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(1) collecting mRNA from a tissue sample to prepare a mRNA-cDNA complementary pair; (2) preparing a double stranded DNA from the mRNA-cDNA complementary pair; (3) fractionating DNA fragments obtained by digesting the double stranded DNA with a restriction enzyme into groups by differences in sequences of two bases adjacent to the restriction enzyme recognition site of each of the DNA fragments, using labeled DNA probes having all possible sixteen combinations of two bases out of A, C, G and T at the 3'"'"' termini thereof; and (4) separating the DNA fragments contained in each of the sixteen groups or complementary strands to the DNA fragments by electrophoresis in terms of length; thereby giving information on the kind and amount of said mRNA.
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21. A method of analysis or assay for nucleotides which comprises:
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(1) collecting mRNA from a tissue sample to prepare a mRNA-cDNA complementary pair; (2) preparing a double stranded DNA from the mRNA-cDNA complementary pair; (3) digesting the double stranded DNA with a restriction enzyme to obtain DNA fragments; (4) discriminating differences in sequences of 1 to 3 bases adjacent to the restriction enzyme recognition sites of the DNA fragments with labeled DNA probes and extending the labeled DNA probes by a complementary strand synthesis to fractionate the DNA fragments into groups, wherein the labeled DNA probes have all possible combinations of 1 to 3 bases out of A, C, G and T at the 3'"'"' termini thereof; and (5) measuring lengths of the DNA fragments which belong to each of said groups, or lengths of the labeled DNA probes extended by said complementary synthesis reaction; wherein based on measured lengths of the DNA fragments fractionated into the groups by the differences in sequences of 1 to 3 bases adjacent to restriction enzyme recognition sites of the DNA fragments, said mRNA is analyzed or assayed thereby giving information on the kind and amount of said mRNA.
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22. A method of analysis or assay for nucleotides which comprises:
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(1) digesting DNA'"'"'s with a restriction enzyme to obtain DNA fragments; (2) discriminating differences in sequences of two bases adjacent to restriction enzyme recognition sites of the DNA fragments with labeled DNA probes and extending the labeled DNA probes by a complementary strand synthesis to fractionate the DNA fragments into sixteen groups, wherein the labeled DNA probes have all possible sixteen combinations of two bases out of A, C, G and T at the 3'"'"' termini thereof; and (3) measuring lengths of the labeled DNA probes extended by said complementary strand synthesis for each group of sixteen groups individually by electrophoresis to obtain an electropherogram for each group of sixteen groups; wherein measured lengths obtained from the sixteen electropherograms are employed as fingerprints.
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23. A method of analysis or assay for nucleotides which comprises:
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(1) digesting a sample DNA with a restriction enzyme to obtain DNA fragments; (2) introducing an oligomer into the DNA fragments at the 3'"'"' termini thereof; (3) dividing a solution containing the DNA fragments into sixteen fractions; (4) adding a different kind of labeled DNA probe to each of the sixteen fractions, respectively, and extending the labeled DNA probes by a complementary strand synthesis, wherein the labeled DNA probes have all possible sixteen combinations of two bases out of A, C, G and T at the 3'"'"' termini thereof; and (5) obtaining sixteen electropherograms by electrophoresis of the labeled DNA probes extended by said complementary strand synthesis in each of the sixteen fractions; wherein each of the sixteen electropherograms is classified by differences in sequences of two bases adjacent to the restriction enzyme recognition sites of the DNA fragments, and the sixteen electropherograms are employed as fingerprints.
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24. A method of analysis or assay for nucleotides which comprises:
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(1) digesting a sample DNA with a restriction enzyme to obtain DNA fragments; (2) introducing a first and a second oligomer having known sequences into the DNA fragments at the 3'"'"' and 5'"'"' termini thereof, respectively; (3) dividing a solution containing the DNA fragments into sixteen first vessels; (4) performing a complementary strand synthesis in each of the sixteen first vessels, respectively, using a different kind of first labeled DNA probes having all possible sixteen combinations of two bases out of A, C, G and T at the 3'"'"' termini thereof, the first labeled DNA probes being complementary to the introduced oligomer or to a part of the recognition sequence recognized by said restriction enzyme, thereby extending the complementary strand of the first labeled DNA probes; (5) removing non-reacted substances in the solutions in each of the sixteen first vessels of step (4), respectively, and, by elevating the temperature of the solution, liberating the DNA fragments from the hybrids of the complementary strand synthesis and recovering the DNA strand extended by the complementary strand synthesis in step (4) in each of the sixteen first vessels; (6) dividing a solution containing the recovered DNA strand extended by the complementary strand synthesis from each of the sixteen first vessels into sixteen second vessels; (7) performing a complementary strand synthesis in each of the sixteen second vessels, respectively, using a different kind of second labeled DNA probes different from the first labeled DNA probes, the second labeled DNA probes having all possible sixteen combinations of two bases out of A, C, G and T at the 3'"'"' termini thereof, the second labeled DNA probes being complementary to the introduced oligomer or to a part of the recognition sequence recognized by said restriction enzyme, thereby extending the complementary strand of the second labeled DNA probes; and (8) obtaining 256 electropherograms by electrophoresis of the second labeled DNA probes extended by said complementary strand synthesis in each of the 256 vessels; wherein each of the 256 electropherograms is classified by differences in sequences of two bases adjacent to the restriction enzyme recognition sites of the DNA fragments, and the 256 electropherograms are employed as fingerprints. - View Dependent Claims (25, 26)
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27. A method of analysis or assay for nucleotides which comprises:
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(1) digesting a sample DNA with a restriction enzyme to obtain DNA fragments; (2) introducing a first and a second oligomer having known sequences into the DNA fragments at the 3'"'"' and 5'"'"' termini thereof, respectively; (3) dividing a solution containing the DNA fragments into sixteen first vessels; (4) performing a complementary strand synthesis in each of the sixteen first vessels, respectively, using different kinds of first labeled DNA probes having all possible sixteen combinations of two bases out of A, C, G and T at the 3'"'"' termini thereof, the first labeled DNA probes being complementary to the introduced oligomer or to a part of the recognition sequence recognized by said restriction enzyme, thereby extending the complementary strand of the first labeled DNA probes; (5) removing non-reacted substances in the solutions in each of the sixteen first vessels of step (4), respectively, and, by elevating the temperature of the solution, liberating the DNA fragments from the hybrids of the complementary strand synthesis and recovering the DNA strand extended by the complementary strand synthesis in step (4), in each of the sixteen first vessels; (6) performing a complementary strand synthesis in a second vessel containing the recovered DNA strand extended by the complementary strand synthesis from each of the sixteen first vessels, using a different kind of second labeled DNA probes different from the first labeled DNA probes, the second labeled DNA probes having all possible sixteen combinations of two bases out of A, C, G and T at the 3'"'"' termini thereof, the second labeled DNA probes being complementary to the introduced oligomer or to a part of the recognition sequence recognized by said restriction enzyme, thereby extending the complementary strand of the second labeled DNA probes, wherein the second labeled DNA probes are labeled with fluorophores having different emission wavelengths; and (7) obtaining 256 electropherograms by electrophoresis of the second labeled DNA probes extended by said complementary strand synthesis in the second vessel; wherein each of the 256 electropherograms is classified by differences in sequences of two bases adjacent to restriction enzyme recognition sites of the DNA fragments, and the 256 electropherograms are employed as fingerprints.
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28. A method of analysis or assay for nucleotides which comprises:
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(1) digesting a sample DNA with a restriction enzyme to obtain DNA fragments; (2) introducing a first and a second oligomer having known sequences into the DNA fragments at the 3'"'"' and 5'"'"' termini thereof, respectively; (3) dividing a solution containing the DNA fragments into sixteen first vessels; (4) performing a complementary strand synthesis in each of the sixteen first vessels, respectively, using a different kind of first labeled DNA probes having all possible sixteen combinations of two bases out of A, C, G and T at the 3'"'"' termini thereof, the first labeled DNA probes being complementary to the introduced oligomer or to a part of the recognition sequence recognized by said restriction enzyme, thereby extending the complementary strand of the first labeled DNA probes; (5) removing non-reacted substances in the solution in each of the sixteen first vessels of step (4), respectively, and, by elevating the temperature of the solution, liberating the DNA fragments from the hybrids of the complementary strand synthesis and recovering the DNA strand extended by the complementary strand synthesis in step (4), in each of the sixteen first vessels; (6) dividing a solution containing the recovered DNA strand extended by the complementary strand synthesis from each of the sixteen first vessels into four second vessels; (7) performing a complementary strand synthesis in each of the second four vessels, using second labeled DNA probes different from the first labeled DNA probes, the second labeled DNA probes having all possible sixteen combinations of two bases out of A, C, G and T at the 3'"'"' termini thereof, the second labeled DNA probes being complementary to the introduced oligomer or to a part of the recognition sequence recognized by said restriction enzyme, thereby extending the complementary strand of the second labeled DNA probes, wherein the second labeled DNA probes are labeled with fluorophores having four different emission wavelengths; and (8) obtaining 256 electropherograms by electrophoresis of the second labeled DNA probes extended by said complementary strand synthesis in the second four vessels;
wherein each of the 256 electropherograms is classified by differences in sequences of two bases adjacent to restriction enzyme recognition sites of the DNA fragments, and the 256 electropherograms are employed as fingerprints.
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Specification