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Direct sequence identification of mutations by cleavage- and ligation-associated mutation-specific sequencing

  • US 5,707,806 A
  • Filed: 06/07/1995
  • Issued: 01/13/1998
  • Est. Priority Date: 06/07/1995
  • Status: Expired due to Term
First Claim
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1. A method for identifying one or more genetic alterations in a target sequence present in a DNA sample, which comprises:

  • a) immobilizing a first DNA sample, said first DNA having a 5'"'"' end and a 3'"'"' end on a solid support under conditions such that said 5'"'"' end is bound to said support and said 3'"'"' end is unbound;

    b) hybridizing said immobilized sample with a second DNA having a 5'"'"' end and a 3'"'"' end wherein said second DNA does not contain the alteration(s), to form heteroduplex DNA containing a mismatch region at the site of an alteration(s);

    c) cleaving one or both strands of said heteroduplex adjacent to said mismatch region to form a gap at or in the vicinity of said alteration;

    d) subjecting said cleaved heteroduplex to conditions of denaturation to dissociate said second DNA and cleaved first DNA 3'"'"' to the site of cleavage from immobilized remaining first DNA;

    e) removing DNA strands cleaved in step c and dissociated in step d from said immobilized remaining first DNA;

    f) ligating a single-stranded oligonucleotide primer of known sequence to the unbound end of said immobilized remaining first DNA to form a ligation product;

    (g) treating said ligation product with a DNA polymerase and an oligonucleotide complementary to said primer of known sequence in the presence of dideoxynucleotides or four nucleotide triphosphates and determining the nucleotide sequence adjacent to the ligated primer sequence; and

    h) comparing said nucleotide sequence with a predetermined cognate wild-type sequence to identify said genetic alteration(s).

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