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Non-competitive co-amplification methods

  • US 6,461,817 B1
  • Filed: 10/20/2000
  • Issued: 10/08/2002
  • Est. Priority Date: 09/12/1997
  • Status: Expired due to Term
First Claim
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1. A non-competitive, quantitative amplification assay for a target DNA sequence, comprising the steps of:

  • providing a reaction mixture for an amplification of said target DNA sequence that includes of a sample suspected to contain said target DNA sequence, reagents for amplifying said target DNA sequence, and a known quantity of a DNA control molecule which has a different sequence from said target sequence, which hybridizes to said target DNA sequence or its complement, and which is co-amplifiable with said target DNA sequence in a single amplification reaction having a single set of reaction kinetics for both the target DNA sequence and the DNA control molecule;

    co-amplifying said control molecule and said target DNA sequence, if present, in said single amplification reaction to produce amplicons of both;

    detecting said amplicons in solution with dual-labeled hybridization probes capable of causing a fluorescent signal to be generated in response to hybridizing to sequences complementary to sequences in the probes, said probes including at least two probes selected from the group consisting of;

    (a) a first probe for said target sequence or its complement but not for said control molecule or its complement;

    (b) a second probe for said control molecule or its complement but not for said target sequence or its complement; and

    (c) and a third probe for both the target sequence or its complement and the control molecule or its complement, wherein binding of said amplicons to said probes is detectable by signals generated by said probes; and

    determining the starting quantity of said target DNA sequence utilizing the ratio of signals from said at least two probes during at least one point during said amplification reaction.

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