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Methods for determining a nucleotide at a specific location within a nucleic acid molecule

  • US 20020142336A1
  • Filed: 02/01/2002
  • Published: 10/03/2002
  • Est. Priority Date: 02/02/2001
  • Status: Abandoned Application
First Claim
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1. A method for detecting the presence or absence of a first nucleotide, at a position within a strand of DNA in a sample, comprising:

  • forming an admixture of a primer and said strand of DNA in said sample and imposing hybridization conditions on said primer and said DNA strand to form a hybridization product, said primer comprising a sequence of DNA which hybridizes with said strand of DNA adjacent to said first nucleotide position and having a second nucleotide opposite said first nucleotide position, said second nucleotide associated with a detectable label, said second nucleotide hybridizing to said first nucleotide in the event said second nucleotide is complementary to said first nucleotide and said second nucleotide not hybridizing to said first nucleotide in the event of said second nucleotide is not complementary;

    applying a proofreading polymerase to the hybridization product under conditions in which said second nucleotide is preferentially excised to form a fluorescently labeled nucleotide product in the event said second nucleotide is not hybridized to said first nucleotide, and in which said second nucleotide is preferentially incorporated into an extension product in the event said second nucleotide is hybridized to said first nucleotide;

    monitoring said sample for the presence of a fluorescent label in association with at least one of said fluorescently labeled nucleotide excision product, said extension product, or said primer using fluorescent polarization, which fluorescent label associated with an excess of said nucleotide excision product is indicative of the absence of said first nucleotide, and which fluorescent label associated with an excess of said extension product is indicative of the presence of said first nucleotide.

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