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Polynucleotide sequencing method

  • US 20040063138A1
  • Filed: 10/10/2003
  • Published: 04/01/2004
  • Est. Priority Date: 02/16/1999
  • Status: Abandoned Application
First Claim
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1. A method for determining a sequence of one or more nucleotides in a target polynucleotide, the method comprising:

  • (a) contacting a target-specific primer with a polynucleotide sample under conditions effective for the primer to anneal specifically to a primer-complementary region in one or more target polynucleotides, to form one or more target-primer hybrid(s), wherein each target-specific primer contains (i) a target binding segment and (ii) a mobility-reducing moiety which does not bind the target, (b) reacting said hybrid(s) with a primer extension reagent in the presence of at least one labeled 3′

    -nucleotide terminator complementary to a selected nucleotide base-type, under conditions effective to form one or more primer extension products which terminate with at least one labeled 3′

    -nucleotide terminator, separating electrophoretically the primer extension product(s) under denaturing conditions in a sieving matrix such that smaller extension products migrate more rapidly than larger extension products, and determining a target sequence on the basis of the mobilities and terminator labels of the separated extension product(s).

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