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Circular probe amplification (CPA) using energy-transfer primers

  • US 7,977,053 B2
  • Filed: 12/12/2006
  • Issued: 07/12/2011
  • Est. Priority Date: 02/27/2001
  • Status: Expired due to Fees
First Claim
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1. A nucleic acid amplification method comprising:

  • (a) providing a closed circular padlock probe molecule;

    a target nucleic acid molecule;

    a forward primer;

    a reverse primer;

    dNTPs; and

    a first DNA polymerase to form a reaction mixture;

    (b) incubating said reaction mixture at about 50°

    C. to about 70°

    C. for 1 minute to 30 minutes;

    (c) creating a multi-tailed complex by cascade rolling circle amplification wherein the multi-tailed complex has multiple primer binding sites, and wherein the multi-tailed complex is not a detectable amplification product;

    (d) activating a second DNA polymerase, wherein said second DNA polymerase is thermostable; and

    (e) thermocycling said multi-tailed complex with said second DNA polymerase, wherein said thermocycling said multi-tailed complex generates a detectable amplification product.

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