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Method and kit for polynucleotide assay including primer-dependant DNA polymerase

  • US 4,851,331 A
  • Filed: 05/16/1986
  • Issued: 07/25/1989
  • Est. Priority Date: 05/16/1986
  • Status: Expired due to Fees
First Claim
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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:

  • (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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