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Assay systems for genetic analysis

  • US 10,131,937 B2
  • Filed: 11/10/2011
  • Issued: 11/20/2018
  • Est. Priority Date: 08/06/2010
  • Status: Active Grant
First Claim
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1. A method for detecting a fetal copy number variation in a maternal plasma or serum sample comprising fetal and maternal cell-free DNA, the method comprising:

  • (a) obtaining the maternal plasma or serum sample comprising fetal and maternal cell-free DNA;

    (b) hybridizing (i) a first set of at least 24 and less than 2000 pairs of fixed sequence oligonucleotides with (ii) the fetal and maternal cell-free DNA in the maternal plasma or serum sample, wherein the percent fetal DNA is less than 25% of the sample, wherein the oligonucleotides of each pair are complementary to regions flanking a non-polymorphic locus within a first chromosome or a portion of the first chromosome, and wherein the melting temperatures (Tms) of first fixed sequence oligonucleotides of each of the pairs of the first set vary in a range of two degrees centigrade;

    (c) hybridizing (i) a second set of at least 24 and less than 2000 pairs of fixed sequence oligonucleotides with (ii) the fetal and maternal cell-free DNA in the maternal plasma or serum sample, wherein the percent fetal DNA is less than 25% of the sample, wherein the oligonucleotides of each pair are complementary to regions flanking a non-polymorphic locus within a second chromosome or a portion of the second chromosome and wherein the Tms of first fixed sequence oligonucleotides of each of the pairs of the second set vary in a range of two degrees centigrade;

    (d) extending the regions between the hybridized oligonucleotides of the first and second sets of fixed sequence oligonucleotide pairs with a polymerase and dNTPs to create hybridized adjacent extended oligonucleotides spanning the non-polymorphic loci in the first and second nucleic acid regions of interest;

    (e) ligating the hybridized adjacent extended oligonucleotides to produce ligation products;

    (f) amplifying the ligation products to produce amplification products, wherein an average number of amplification products per locus is greater than 100;

    (g) detecting the amplification products at least an average of 100 times to count the relative frequency of each non-polymorphic locus within the first and second nucleic acid regions of interest; and

    (h) detecting a fetal copy number variation of the first nucleic acid region of interest in the maternal serum or plasma sample relative to the second nucleic acid region of interest.

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