Method for determining nucleotide identity through primer extension

  • US 5,888,819 A
  • Filed: 03/05/1991
  • Issued: 03/30/1999
  • Est. Priority Date: 03/05/1991
  • Status: Expired due to Term
First Claim
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1. A method of determining the identity of a nucleotide base at a specific position in a nucleic acid of interest, which comprises:

  • (a) treating a sample containing the nucleic acid of interest, if the nucleic acid is double-stranded, so as to obtain unpaired nucleotide bases spanning the specific position, or directly employing step (b) if the nucleic acid of interest is single-stranded;

    (b) contacting the sample from step (a), with an oligonucleotide primer which is fully complementary to and which hybridizes specifically to a stretch of nucleotide bases present in the nucleic acid of interest immediately adjacent to the nucleotide base to be identified, under high stringency hybridization conditions, so as to form a duplex between the primer and the nucleic acid of interest such that the nucleotide base to be identified is the first unpaired base in the template immediately downstream of the 3'"'"' end of the primer in said duplex; and

    (c) contacting the duplex from step (b), in the absence of dATP, dCTP, dGTP, or dTTP, with at least two different terminators of a nucleic acid template-dependent, primer extension reaction capable of specifically terminating the extension reaction in a manner strictly dependent upon the identity of the unpaired nucleotide base in the template immediately downstream of the 3'"'"' end of the primer wherein one of said terminators is complementary to said nucleotide base to be identified and wherein at least one of said terminators is labeled with a detectable marker;

    wherein said contacting is under conditions sufficient to permit base pairing of said complementary terminator with the nucleotide base to be identified and occurrence of a template-dependent primer extension reaction sufficient to incorporate said complementary terminator onto the 3'"'"' end of the primer to thereby extend said 3'"'"' end of said primer by one terminator;

    (d) determining the presence and identity of the nucleotide base at the specific position in the nucleic acid of interest by detecting the detectable marker of said incorporated terminator while said terminator is incorporated at the 3'"'"' end of the extended primer, and wherein said detection is conducted in the absence of non-terminator nucleotides.

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