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Asymmetric PCR with nuclease-free polymerase or nuclease-resistant molecular beacons

  • US 20030134307A1
  • Filed: 10/25/2002
  • Published: 07/17/2003
  • Est. Priority Date: 10/25/2001
  • Status: Abandoned Application
First Claim
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1. A method for performing combined amplification and detection of a nucleic acid target, the method comprising:

  • providing a molecular beacon comprising a region of complementarity to a first region of a first strand of the nucleic acid target;

    providing a first primer comprising a region of identity with a second region of the first strand of the nucleic acid target;

    providing a second primer comprising a region of complementarity to a third region of the first strand of the nucleic acid target, the third region being 3′

    of the first region, and the first region being 3′

    of the second region;

    wherein the first primer is provided at a concentration that is at least about 1.3 times that of the second primer;

    providing a template nucleic acid comprising the first strand of the nucleic acid target, a second strand of the nucleic acid target that is complementary to the first strand, or both;

    providing a polymerase substantially lacking 5′

    to 3′

    nuclease activity;

    amplifying the target nucleic acid by subjecting the template nucleic acid, the first and second primers, the molecular beacon, and the polymerase to cycles comprising denaturation, annealing, and extension steps; and

    , detecting a signal from the molecular beacon at at least one time point during or after the cycles.

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