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Methods for determining sequences information in polynucleotides using mass spectrometry

  • US 5,885,775 A
  • Filed: 10/04/1996
  • Issued: 03/23/1999
  • Est. Priority Date: 10/04/1996
  • Status: Expired due to Term
First Claim
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1. A method for identifying a putative point mutation in a polynucleotide using mass spectrometry, comprising the steps of:

  • a. providing a primer having a sequence that hybridizes adjacent said putative point mutation of said single strand of said polynucleotide;

    b. heating said polynucleotide and said primer to a temperature sufficient to melt said polynucleotide; and

    c. cooling said melted polynucleotide and said primer to a temperature sufficient to hybridize said primer to said single strand of said polynucleotide, thereby forming a duplex wherein the 3'"'"'-end of said primer is hybridized adjacent the putative point mutation of said single strand of said polynucleotide;

    d. extending the 3'"'"'-end of said primer by adding a single dideoxynucleotide base complementary to the putative point mutation of the polynucleotide, wherein said dideoxynucleotide base has a mass range of about 273 to about 313,using a polynucleotide thermostable polymerase having substantially the same relative rate of incorporation of dideoxynucleotide bases as deoxynucleotide bases, thereby forming an extended primer; and

    e. analyzing the extended primer using mass spectrometry to determine the identity of the dideoxynucleotide base at the putative point mutationwherein steps a-d are repeated a sufficient number of times to permit detection of said extended primer by mass spectrometry.

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