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Detection of a nucleic acid sequence or a change therein

  • US 5,856,092 A
  • Filed: 04/27/1995
  • Issued: 01/05/1999
  • Est. Priority Date: 02/13/1989
  • Status: Expired due to Term
First Claim
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1. A method for detecting whether a specific nucleotide or base is at a particular position in a specific polynucleotide sequence comprising:

  • a) exposing, under hybridizing conditions, said specific polynucleotide sequence to an oligonucleotide primer wherein said primer has a sequence complementary to part of the specific polynucleotide sequence wherein said primer has incorporated at its 5'"'"' end an element selected from the group consisting of a separation element and a detectable element, and wherein said primer hybridizes at a location selected from the group consisting of (i) immediately adjacent to the particular position and (ii) not immediately adjacent to the particular position whereby there is an intervening sequence between the particular position and primer bound to the specific polynucleotide sequence;

    b) extending said hybridized primer up to and including said specific nucleotide or base wherein the 3'"'"' terminal nucleotide is a ddNTP and further includes an element selected from the group consisting of a separation element and a detectable element with the proviso that said extended primer has at least one separation element and at least one detectable element;

    c) separating the product of step b) into fractions wherein one said fraction contains the primer extension product that contains the chain terminating nucleotide at said particular position; and

    d) determining whether said primer extension product having said chain terminating nucleotide at said particular position is present in said fraction by assaying said fraction wherein the assay does not include a digestion step.

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