METHODS FOR PROCESSING DNA SUBSTRATES

  • US 20160362726A1
  • Filed: 08/31/2016
  • Published: 12/15/2016
  • Est. Priority Date: 01/31/2014
  • Status: Active Grant
First Claim
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1. A method of multiplex PCR-based enrichment of a target substrate comprising the steps of:

  • (i) introducing a plurality of polymerase chain reaction components into a sample comprising a nucleic acid substrate to provide a PCR reaction mixture, wherein the plurality of polymerase chain reaction components comprise a plurality of different target-specific primer pairs for amplifying a plurality of different loci of the nucleic acid substrate, a universal primer, a DNA polymerase, and dNTPs, wherein each of the plurality of different target-specific primer pairs comprise a forward primer and a reverse primer, wherein the forward primer and the reverse primer comprise a 3′

    complementary sequence that is complementary to a first sequence of the nucleic acid substrate and a second sequence of the nucleic acid substrate, respectively, wherein the first sequence and second sequence is different for each of the plurality of different target-specific primer pairs, and wherein the forward primer and the reverse primer of each of the plurality of different target-specific primer pairs further comprise a 5′

    terminal sequence that is not complementary to the nucleic acid substrate, wherein the 5′

    terminal sequence comprises a universal adaptor sequence, wherein the universal primer comprises the universal adaptor sequence and a modified base that targets cleavage of the universal primer by an endonuclease, wherein the universal primer is at a final concentration in the PCR reaction mixture that is in excess of the final concentration of each of the plurality of different target-specific primer pairs;

    (ii) exposing the PCR reaction mixture to at least two initial PCR cycles comprising a first annealing temperature for a first annealing duration to generate a plurality of different target specific amplicons, wherein each of the plurality of different target specific amplicons comprise the 5′

    terminal sequence at their 5′

    terminal regions;

    (iii) exposing the PCR reaction mixture to an appropriate number of additional PCR cycles to further amplify the plurality of different target specific amplicons, wherein the additional PCR cycles comprise the first annealing temperature for a second annealing duration, wherein the first annealing duration is greater than the second annealing duration.

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