×

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

  • US 7,556,924 B2
  • Filed: 10/31/2007
  • Issued: 07/07/2009
  • Est. Priority Date: 05/29/1996
  • Status: Expired due to Fees
First Claim
Patent Images

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 padlock oligonucleotide probes, each probe comprising (a) a first target-specific portion and a 5′

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

    downstream primer-specific portion, wherein the first and second target-specific portions of a particular probe 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 target-specific portions of a particular probe are hybridized to any other nucleotide sequence present in the sample;

    providing a ligase;

    blending the sample, the one or more padlock oligonucleotide probes, 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 padlock oligonucleotide probe is present in the sample;

    providing one or a plurality of oligonucleotide primer sets, each set comprising (a) an upstream primer and (b) a downstream primer;

    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 identity one or more target nucleotide sequences in the sample.

View all claims
  • 2 Assignments
Timeline View
Assignment View
    ×
    ×