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Processes and kits for identifying aneuploidy

  • US 9,926,593 B2
  • Filed: 12/20/2010
  • Issued: 03/27/2018
  • Est. Priority Date: 12/22/2009
  • Status: Active Grant
First Claim
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1. A multiplex method for identifying the presence or absence of an aneuploidy of a target chromosome in a sample from a pregnant female subject, which comprises:

  • a. providing about 20 to about 56 amplification primer pairs, wherein each amplification primer pair specifically hybridizes with polynucleotides of a nucleotide sequence species set, wherein;

    i) the polynucleotides of a nucleotide sequence species set are present on two or more different chromosomes at different loci, comprising a target chromosome and one or more reference chromosomes not associated with the aneuploidy;

    (ii) the polynucleotides of a nucleotide sequence species set differ by one or more mismatch nucleotides;

    (iii) the polynucleotides of a nucleotide sequence species set are reproducibly amplified by a single pair of amplification primers relative to each other;

    (iv) each amplified polynucleotide in a nucleotide sequence species set comprises a nucleotide sequence having the one or more mismatch nucleotides and (v) the polynucleotides of the nucleotide sequence species sets have nucleotide sequences corresponding to SEQ ID NOs;

    4736, 4808, 4258, 4413, 4537, 4692, 4203, 4077, 4697, 4436, 3869, 4475, 4078, 4396, 4782, 4683, 4279, 3940, 4236, 3865, 4733, 4168, 3818, 4563, 4906, 3807, 3849, 4594, 4022, 4538, 4219, 4548, 4382, 4374, 4444, 4305, 4403, 4307, 4059, 4767, 3994, 4561, 4209, 3850, 4686, 3985, 4608, 3963, 4038, 4948, 4468, 4879, 4196, 3967, 4150 and 4102;

    b. contacting in one or more reaction vessels under amplification conditions, extracellular nucleic acid of the sample comprising fetally derived and maternally derived nucleic acid with amplification primer pairs consisting of the about 20 to about 56 amplification primer pairs of (a), wherein about 10 to about 25 of the amplification primer pairs are in each of the one or more reaction vessels, and each primer pair amplifies only a single nucleotide species set in the reaction vessel, thereby producing about 20 to about 56 species sets of amplified polynucleotides;

    c. determining the amount of each amplified polynucleotide in each set by detecting the one or more mismatch nucleotides in each amplified polynucleotide by primer extension, sequencing, Q-PCR or mass spectrometry;

    d. determining a ratio between the relative amount of (i) an amplified target polynucleotide and (ii) an amplified reference polynucleotide, for each set; and

    e. identifying the presence or absence of an aneuploidy of a target chromosome based on the ratios from the about 20 to about 56 species sets of amplified polynucleotides.

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