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Determination of nucleotide sequence variations through limited primer extension

  • US 6,355,433 B1
  • Filed: 06/02/2000
  • Issued: 03/12/2002
  • Est. Priority Date: 06/02/2000
  • Status: Expired due to Fees
First Claim
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1. A method of analyzing a variant site of a target nucleic acid, comprising(a) conducting a template-dependent extension reaction comprising extending a primer in the presence of the target nucleic acid and a mixture of nucleotides comprising at least one labeled extendible nucleotide and at least one labeled non-extendible nucleotide, each labeled extendible nucleotide and labeled non-extendible nucleotide being complementary to a different allelic form of the target nucleic acid and optionally differentially labeled, wherein the primer hybridizes to a segment of the target nucleic acid such that the 3′

  • -end of the primer hybridizes adjacent the variant site of the target nucleic acid, whereby if the labeled extendible nuclcotide is complementary to the nucleotide occupying the variant site, the primer is extended by incorporation of the labeled extendible nucleotide, and can be extended further if one or more nucleotides downstream of the variant site are complementary to one of the nucleotides in the mixture, and if the labeled non-extendible nucleotide is complementary to the nucleotide occupying the variant site, the primer is extended by incorporation of the labeled non-extendible nucleotide; and

    (b) detecting incorporation of labeled nucleotide into the extended primer, the identity of the labeled nucleotide incorporated into the primer indicating the identity of the nucleotide at the variant site, wherein the identity of the incorporated nucleotide is determined from the label borne by the incorporated nucleotide and/or the size of the extended primer.

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