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Amplification method for polynucleotide detection assays

  • US 5,397,698 A
  • Filed: 11/02/1993
  • Issued: 03/14/1995
  • Est. Priority Date: 07/23/1987
  • Status: Expired due to Term
First Claim
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1. A method of determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises:

  • (a) forming in the presence of nucleoside triphosphates and template-dependent polynucleotide polymerase an extension of a polynucleotide analyte having a 3'"'"' hydroxy nucleotide, a part thereof including said 3'"'"' hydroxy nucleotide being hybridized with a binding polynucleotide sequence of a single stranded pattern polynucleotide comprising said binding polynucleotide sequence connected at its 5'"'"' end to two or more template sequences each containing one or more site specific cleavage sequences,(b) cleaving into fragments said extension at cleavable polynucleotide sequences in the presence of means for specifically cleaving said cleavable polynucleotide sequences when said extension is hybridized with said site specific cleavage sequences,(c) dissociating said fragments,(d) hybridizing said fragments with said single stranded pattern polynucleotide,(e) forming in the presence of said nucleoside triphosphates and said template dependent polynucleotide polymerase an extension of said fragments hybridized with said single stranded pattern polynucleotide,(f) repeating steps (b)-(e) above wherein steps (b)-(e) are conducted simultaneously or wholly or partially sequentially, and(g) detecting said fragments or fragments complementary thereto, the presence thereof indicating the presence of said polynucleotide analyte in said sample.

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