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Methods for simultaneous amplification of target loci

  • US 10,597,723 B2
  • Filed: 02/27/2019
  • Issued: 03/24/2020
  • Est. Priority Date: 05/18/2010
  • Status: Active Grant
First Claim
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1. A method of amplifying target loci in a nucleic acid sample, the method comprising:

  • (a) contacting a nucleic acid sample comprising target loci with a library of test primers comprising at least 1,000 different primer pairs to produce a reaction mixture in one reaction volume;

    wherein each primer pair includes a forward test primer and a reverse test primer that hybridize to the same target locus, and wherein the primers do not include molecular inversion probes (MIPs);

    (b) subjecting the reaction mixture to PCR conditions to produce amplified products comprising target amplicons;

    wherein the concentration of each test primer is less than 10 nM;

    wherein the length of the annealing step of the reaction conditions is greater than 10 minutes;

    wherein at least 1,000 different target loci are simultaneously amplified; and

    wherein (i) less than 20% of the amplified products are test primer dimers, (ii) at least 80% of the amplified products are target amplicons, and (iii) at least 80% of the target loci are amplified; and

    (c) sequencing the amplified products.

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