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Methods and systems for identifying nucleotides by primer extension

  • US 6,720,148 B1
  • Filed: 02/20/2002
  • Issued: 04/13/2004
  • Est. Priority Date: 02/22/2001
  • Status: Expired due to Term
First Claim
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1. A method of identifying a nucleotide in at least a first position in a polynucleotide sequence, comprising:

  • providing a polynucleotide target sequence;

    hybridizing the target sequence with a first oligonucleotide probe, wherein;

    the probe comprises a first subsequence of nucleotides, a first 3′

    -terminal nucleotide, and a first florescent label coupled to the 3′

    -terminal nucleotide;

    the subsequence is complementary to a portion of the target sequence that is immediately adjacent to the first position; and

    the 3′

    -terminal nucleotide is complementary to one possible nucleotide in the first position;

    contacting the hybridized probe and target sequence with polymerase extension reagents in a first extension reaction mixture;

    measuring a level of polarized fluorescence emitted from the first extension reaction mixture, a decrease in polarized fluorescence indicating the presence of polymerase extension of the probe, the presence of polymerase extension of the probe indicating that the 3′

    -terminal nucleotide is complementary to the nucleotide in the first position; and

    identifying the nucleotide in the first position.

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