Method and kit for polynucleotide assay including primer-dependant DNA polymerase
First Claim
1. A method for the determination of a target nucleotide sequence in the nucleic acid of a biological sample which comprises the steps:
- (a) contacting a biological sample having a target nucleotide sequence unseparated from plural non-target polynucleotide sequences with a probe polynucleotide strand under conditions sufficient for the probe polynucleotide to bind only to the target nucleotide sequence to form a hybrid having a double-stranded portion including the 3'"'"' end of the probe polynucleotide, with the target nucleic acid sequence extending in a 3'"'"' to 5'"'"' direction beyond the 3'"'"' end nucleotide of the probe polynucleotide;
(b) extending the probe polynucleotide strand of the hybrid beyond its 3'"'"' end in the 5'"'"' to 3'"'"' direction by using the target nucleic acid strand as a template strand and incorporating nucleoside triphosphates from solution, a plurality of the nucleotides incorporated into the extended probe strand being detectably-modified nucleotides; and
(c) determining the amount of detectably-modified nucleotides which have been incorporated into probe polynucleotide strand as a measure of target nucleotide sequence in the biological sample.
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Accused Products
Abstract
A probe polynucleotide binds to a target nucleotide sequence in the nucleic acid of a biological sample, and then is enzymatically extended in the 3'"'"'-direction with a mixture of nucleoside triphosphates including at least one nucleoside triphosphate that has been detectably labeled. After separating extended hybrid from unreacted nucleoside triphosphates, detectably-modified nucleotides which have been incorporated are determined. In some forms, the 3'"'"'-terminal nucleotide of the probe polynucleotide is selected to form a matched pair with some sample strands, but a mismatched pair with other sample strands. In such cases, if the primer dependent enzyme used for extension is one lacking 3'"'"'-exonuclease activity, then only those hybrids forming such a matched pair will be extended and subsequently determined.
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Citations
24 Claims
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1. A method for the determination of a target nucleotide sequence in the nucleic acid of a biological sample which comprises the steps:
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(a) contacting a biological sample having a target nucleotide sequence unseparated from plural non-target polynucleotide sequences with a probe polynucleotide strand under conditions sufficient for the probe polynucleotide to bind only to the target nucleotide sequence to form a hybrid having a double-stranded portion including the 3'"'"' end of the probe polynucleotide, with the target nucleic acid sequence extending in a 3'"'"' to 5'"'"' direction beyond the 3'"'"' end nucleotide of the probe polynucleotide; (b) extending the probe polynucleotide strand of the hybrid beyond its 3'"'"' end in the 5'"'"' to 3'"'"' direction by using the target nucleic acid strand as a template strand and incorporating nucleoside triphosphates from solution, a plurality of the nucleotides incorporated into the extended probe strand being detectably-modified nucleotides; and (c) determining the amount of detectably-modified nucleotides which have been incorporated into probe polynucleotide strand as a measure of target nucleotide sequence in the biological sample. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A kit for the determination of a target nucleotide sequence in the nucleic acid of a biological sample comprising:
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(a) a probe polynucleotide comprising a primer sequence substantially complementary to target and non-target nucleotide sequences in a biological sample, wherein said target nucleotide sequence contains a nucleotide complementary to the 3'"'"'-terminal nucleotide of said probe to form a template for primer-dependent nucleic acid polymerase and wherein said non-target nucleotide sequences contain nucleotides not complementary to said 3'"'"'-terminal nucleotide of said probe; (b) a plurality of nucleoside triphosphates, wherein at least one of said nucleoside triphosphates is modified to contain a detectable label; and (c) a primer-dependent nucleic acid polymerase which is substantially free of exonuclease activity, wherein said polymerase extends primers in a 5'"'"' to 3'"'"' direction when the 3'"'"'-terminal nucleotide of the primer is complementary to a nucleotide on a template polynucleotide sequence. - View Dependent Claims (22, 23, 24)
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Specification