Nucleic acid sequence analysis with nucleoside-5'-O-(1-thiotriphosphates)
First Claim
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1. A method of determining the nucleotide sequence of a target nucleic acid, the method comprising the steps of:
- providing a template nucleic acid, the template nucleic acid containing the target nucleic acid;
providing an oligonucleotide primer for the template nucleic acid;
hybridizing the oligonucleotide primer to the template nucleic acid;
extending the oligonucleotide primer with a nucleic acid polymerase in a reaction mixture comprising nucleotide precursors and at least one chain-terminating nucleotide to form a nested series of DNA fragment populations such that every DNA fragment of the same size terminates with the same chain-terminating nucleotide, wherein said nucleotide precursors consist of nucleoside-5'"'"'-O-(1-thiotriphosphate)s;
separating the DNA fragment populations by size; and
identifying the chain-terminating nucleotide associated with each DNA fragment population.
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Abstract
A chain-termination method of nucleic acid sequence determination is provided wherein nucleoside triphosphate precursors are replaced with their 1-thiotriphosphate analogs in the polymerization step. This substitution results in more uniform bands of electrophoretically separated DNA fragments which, in turn, results in more accurate base determination.
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Citations
20 Claims
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1. A method of determining the nucleotide sequence of a target nucleic acid, the method comprising the steps of:
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providing a template nucleic acid, the template nucleic acid containing the target nucleic acid; providing an oligonucleotide primer for the template nucleic acid; hybridizing the oligonucleotide primer to the template nucleic acid; extending the oligonucleotide primer with a nucleic acid polymerase in a reaction mixture comprising nucleotide precursors and at least one chain-terminating nucleotide to form a nested series of DNA fragment populations such that every DNA fragment of the same size terminates with the same chain-terminating nucleotide, wherein said nucleotide precursors consist of nucleoside-5'"'"'-O-(1-thiotriphosphate)s; separating the DNA fragment populations by size; and identifying the chain-terminating nucleotide associated with each DNA fragment population. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. In a method of determining the nucleotide sequence of a target nucleic acid, the method of the type wherein a nested series of DNA fragment populations is generated by extending an oligonucleotide primer with a nucleic acid polymerase in the presence of one or more chain-terminating nucleotides and wherein the nested series of DNA fragment populations is separated by size, an improvement comprising:
extending the oligonucleotide primer with the nucleic acid polymerase in a reaction mixture comprising nucleotide precursors consisting of nucleoside-5'"'"'-O-(1-thiotriphosphate)s. - View Dependent Claims (18, 19, 20)
Specification