×

Protection of barcodes during DNA amplification using molecular hairpins

  • US 10,557,134 B2
  • Filed: 02/24/2016
  • Issued: 02/11/2020
  • Est. Priority Date: 02/24/2015
  • Status: Active Grant
First Claim
Patent Images

1. A method of amplifying a target nucleic acid in a sample comprising:

  • a. contacting a sample comprising a target nucleic acid with a target-specific hairpin barcode forward primer and a target-specific reverse primer,wherein the hairpin barcode forward primer comprises, in a 5′

    to 3′

    direction;

    a 5′

    stem sequence, an adaptor sequence, a barcode sequence, a 3′

    stem sequence, and a 3′

    target-specific sequence, wherein the 5′

    stem sequence and the 3′

    stem sequence each comprise sequence complementary to each other and the complementary sequences hybridize to each other under a closed annealing temperature and do not hybridize to each other at an open annealing temperature;

    b. amplifying the target nucleic acid by performing 2-5 cycles of PCR pre-amplification on the target nucleic acid, wherein the 2-5 cycles of PCR pre-amplification have an annealing temperature less than or equal to the closed annealing temperature of the hairpin barcode forward primer, to generate a plurality of pre-amplification target nucleic acids;

    c. contacting the plurality of pre-amplification target nucleic acids with an adaptor-specific forward primer and an adaptor-specific reverse primer; and

    d. amplifying the pre-amplification target nucleic acid by performing at least 10 cycles of PCR amplification on the pre-amplification target nucleic acids, wherein at least 3 of the at least 10 cycles of PCR-based amplification have an annealing temperature greater than or equal to the open annealing temperature of the hairpin barcode forward primer, to generate a plurality of target nucleic acid amplicons, wherein the target nucleic acid amplicons comprise the adaptor sequence and the barcode sequence.

View all claims
  • 3 Assignments
Timeline View
Assignment View
    ×
    ×