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Assay systems for genetic analysis

  • US 10,131,951 B2
  • Filed: 07/08/2016
  • Issued: 11/20/2018
  • Est. Priority Date: 08/06/2010
  • Status: Active Grant
First Claim
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1. A method of detecting a presence or absence of copy number variation (CNV) in a mixed sample from a single subject, said mixed sample comprising nucleic acids from at least two sources comprising a major source and a minor source, the method comprising:

  • a) hybridizing at least 24 first sets of two fixed sequence oligonucleotides to the nucleic acids in the mixed sample, wherein each first set of two fixed sequence oligonucleotides is complementary to a locus in a first region of interest, each first set of two fixed sequence oligonucleotides comprising universal primer regions, and wherein melting temperatures (Tms) of first fixed sequence oligonucleotides of each first set of two fixed sequence oligonucleotides vary in a range of two degrees centigrade;

    b) hybridizing at least 24 second sets of two fixed sequence oligonucleotides to the nucleic acids in the mixed sample, wherein each second set of two fixed sequence oligonucleotides is complementary to a locus in a second region of interest, each second set of two fixed sequence oligonucleotides comprising universal primer regions, and wherein Tms of first fixed sequence oligonucleotides of each second set of two fixed sequence oligonucleotides vary in a range of two degrees centigrade;

    c) extending one of said hybridized fixed sequence oligonucleotides of each first set of two fixed sequence oligonucleotides with a polymerase between the hybridized oligonucleotides of each first set of two fixed sequence oligonucleotides to produce adjacently hybridized fixed sequence oligonucleotides and extending one of said hybridized fixed sequence oligonucleotides of each second set of two fixed sequence oligonucleotides with a polymerase between the hybridized oligonucleotides of each second set of two fixed sequence oligonucleotides to produce adjacently hybridized fixed sequence oligonucleotides;

    d) ligating the adjacently hybridized fixed sequence oligonucleotides of each first set of two fixed sequence oligonucleotides to create contiguous ligation products and ligating the adjacently hybridized fixed sequence oligonucleotides of each second set of two fixed sequence oligonucleotides to create contiguous ligation products;

    e) amplifying the contiguous ligation products using the universal primer regions on each first and second sets of two fixed sequence oligonucleotides to produce amplification products;

    f) detecting the amplification products; and

    g) detecting a copy number variation of the first region of interest relative to the second region of interest.

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