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

  • US 7,097,980 B2
  • Filed: 05/24/2004
  • Issued: 08/29/2006
  • Est. Priority Date: 05/29/1996
  • Status: Expired due to Fees
First Claim
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1. A method for identifying two or more of a plurality of sequences differing by one or more single-base changes, insertions, deletions, or translocations in a plurality of target nucleotide sequences comprising:

  • providing a sample potentially containing one or more target nucleotide sequences with a plurality of sequence differences;

    providing one or more primary oligonucleotide primer groups, each group comprised of one or more primary oligonucleotide primer sets, each set characterized by (a) a first oligonucleotide primer, having a target-specific portion and a 5′

    upstream secondary primer-specific portion, and (b) a second oligonucleotide primer, having a target-specific portion and a 5′

    upstream secondary primer-specific portion, wherein the first oligonucleotide primers of each set in the same group contain the same 5′

    upstream secondary primer-specific portion and the second oligonucleotide primers of each set in the same group contain the same 5′

    upstream secondary primer-specific portion;

    providing a polymerase;

    blending the sample, the primary oligonucleotide primers, and the polymerase to form a primary polymerase chain reaction mixture;

    subjecting the primary polymerase chain reaction mixture to two or more polymerase chain reaction cycles to form primary extension products complementary to the target nucleotide sequence;

    providing one or a plurality of secondary oligonucleotide primer sets, each set characterized by (a) a first secondary primer containing the same sequence as the 5′

    upstream portion of a first primary oligonucleotide primer, and (b) a second secondary primer containing the same sequence as the 5′

    upstream portion of a second primary oligonucleotide primer from the same primary oligonucleotide primer set as the first primary oligonucleotide contained by the first secondary primer, wherein a set of secondary oligonucleotide primers may be used to amplify all of the primary extension products in a given group;

    blending the primary extension products, the secondary oligonucleotide primers, and a polymerase to form a secondary polymerase chain reaction mixture;

    subjecting the secondary polymerase chain reaction mixture to two or more polymerase chain reaction cycles to form secondary extension products complementary to the primary extension products;

    providing a plurality of oligonucleotide probe sets, each set characterized by (a) a first oligonucleotide probe, having a secondary extension product-specific portion and a detectable reporter label, and (b) a second oligonucleotide probe, having a secondary extension product-specific portion;

    providing a ligase;

    blending the secondary extension products, the plurality of 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 detectable reporter label and (b) the secondary extension product-specific portions; and

    detecting the reporter labels of the ligation product sequences, thereby indicating the presence of two or more target nucleotide sequences in the sample.

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