Detection of nucleic acid sequence differences using coupled ligase detection and polymerase chain reactions

CAFC
  • US 6,797,470 B2
  • Filed: 07/30/2001
  • Issued: 09/28/2004
  • Est. Priority Date: 05/29/1996
  • Status: Expired due to Term
First Claim
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1. A method for identifying one or more different target nucleotide sequences comprising:

  • providing a sample potentially containing one or more target nucleotide sequences comprising sequence differences;

    providing one or more oligonucleotide probe sets, each set comprising (a) a first oligonucleotide probe comprising a target-specific portion and a 5′

    upstream primer-specific portion and (b) a second oligonucleotide probe comprising a target-specific portion and a 3′

    downstream primer-specific portion, wherein the first and second oligonucleotide probes in each particular set are suitable for ligation together when hybridized on a corresponding target nucleotide sequence, but have a mismatch which interferes with such ligation when first and second oligonucleotide probes are hybridized to any other nucleotide sequence present in the sample;

    providing a ligase;

    blending the sample, the one or more oligonucleotide probe sets, and the ligase to form a ligase detection reaction mixture;

    subjecting the ligase detection reaction mixture to one or more ligase detection reaction cycles to form a ligation product sequence comprising (a) the 5′

    upstream primer specific portion, (b) the target-specific portions, and (c) the 3′

    downstream primer-specific portion, when the respective target nucleotide sequence of the corresponding oligonucleotide probe set is present in the sample;

    providing one or a plurality of oligonucleotide primer sets, each set comprising (a) an upstream primer containing the same sequence as the 5′

    upstream primer-specific portion of the ligation product sequence and (b) a downstream primer complementary to the 3′

    downstream primer-specific portion of the ligation product sequence;

    providing a polymerase;

    blending the ligase detection reaction mixture with the one or a plurality of oligonucleotide primer sets, and the polymerase to form a polymerase chain reaction mixture;

    subjecting the polymerase chain reaction mixture to one or more polymerase chain reaction cycles to form extension products comprising the ligation product sequence and/or complements thereof; and

    detecting the extension products to identify one or more target nucleotide sequences in the sample.

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