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Global amplification using a randomly primed composite primer

  • US 9,175,325 B2
  • Filed: 06/14/2013
  • Issued: 11/03/2015
  • Est. Priority Date: 04/14/2003
  • Status: Active Grant
First Claim
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1. A method for amplification of a template DNA, comprising:

  • (a) incubating the template DNA with(i) a pool of first composite primers, each first composite primer comprising(1) a 5′

    RNA portion that is not hybridizable to the template DNA and(2) a 3′

    DNA portion, wherein the 3′

    DNA portion of each of the first composite primers comprises a random sequence that is different from the 3′

    DNA portion of the other first composite primers in the pool, and(ii) a DNA polymerase having strand displacement activity, whereby a complex comprising an RNA/DNA heteroduplex is generated;

    (b) incubating the complex under conditions that permit cleavage of RNA in the RNA/DNA heteroduplex; and

    (c) incubating the complex with an amplification primer, wherein the amplification primer is a composite primer comprising a 5′

    RNA portion and a 3′

    DNA portion, wherein the amplification primer anneals to sequence complementary to the RNA sequence cleaved from the RNA/DNA heteroduplex, whereby multiple copies of an amplification product are generated.

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