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Accelerating identification of single nucleotide polymorphisms and alignment of clones in genomic sequencing

  • US 6,534,293 B1
  • Filed: 01/05/2000
  • Issued: 03/18/2003
  • Est. Priority Date: 01/06/1999
  • Status: Expired due to Term
First Claim
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1. A method for large scale detection of single nucleotide plymorphisms on a DNA array comprising:

  • creating a repsentation of the genome from a clinical sample, wherein said creating comprises;

    subjecting the clinical sample to treatment with a first restriction endonuclease under conditions effective to cleave DNA so that a first non-palindromic overhang is created in the clinical sample;

    subjecting the clinical sample to treatment with one or more second restriction endonuclease under conditions effective to cleave DNA so that a second overhang is created in the clinical sample;

    adding complementary linker adapters to the first overhang and complementary linker adpaters to the second overhang, in the presence of a ligase, the first restriction endonuclease, and the one or more second restriction endonuclease to bind the complementary linker adapters, respectively, to the first and second overhangs; and

    adding PCR primers “

    and amplifying fragments from the restriction endonuclease treatment to generate”

    a representation of the genome in the form of nucleic acid target sequences;

    providing a plurality of oligonucleotide probe sets, each set characterized by (a) a frist oligonucleotide probe, having a target-specific portion and an addressable array-specific portion, and (b) a second oligonuclotide probe, having a target-specific portion and a detectable reporter label, wherein the oligonueotide probes in a particular set are suitable for ligation together when hybridized adjacent to one another on a corresponding target sequence, but have a mismatch which interferes with such ligation when hybridized to any other nucleic acid sequence present in the representation;

    providing a ligase;

    blending the representation of the genome in the form of nucleic acid target sequences, the plurality of oligonucleotide probe sets, and the ligase to form a mixture;

    subjecting the mixture to one or more ligase detection reaction cycles comprising a denaturation treatemtn, wherein any hybridized oligonucleotides are seprated from the target sequences by heating, and hybridization treatment, wherein the oligonucleotide probe sets hybridize at adjacent positions in a base-specific manner to their respective target sequences, if present in the sample, and ligate to one another at their junction to form a ligated product sequence containing (a) the addressable array-specific portin, (b) the target-specific portions connected together, and (c) the detectable reporter label, and, wherein the oligonucleotide probe sets may hybridize to nucleic acid sequences in the representation of the genome other than their respective target sequences but do not ligate together due to a presence of noe or more mismatches proximate their junction and individually separate during the denaturation treatement;

    providing a support with different capture oligonucleotides immobilized at particular sites, wherein the capture oligonucleotides have nucleotide sequences complementary to the addressable array-specific portions;

    contacting the mixture, after said subjecting, with the support under cinditions effective to hybridize the addressable array-specific portions to the capture oligonucleotides in a base-specific manner, thereby capturing the addressable array-specific portions on the support at the site with the complementary capture oligonucleotide; and

    detecting the reporter lables of ligated product sequences captured on the support at particular sites, therby indicating the presence of single nucleotide polymorphisms.

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