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Use of mass labelled probes to detect target nucleic acids using mass spectrometry

  • US 7,618,780 B2
  • Filed: 05/19/2005
  • Issued: 11/17/2009
  • Est. Priority Date: 05/20/2004
  • Status: Expired due to Fees
First Claim
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1. A method of detecting the presence of a target nucleic acid in a sample, which method comprisesa) contacting the sample, under hybridizing conditions, with a probe for said target nucleic acid, wherein said probe comprises two terminal nucleic acid target recognition sequences that are complementary to and capable of hybridizing to two neighbouring regions of the target sequence, and wherein the probe further comprises a probe identification sequence and a pair of primer binding sequences;

  • b) covalently connecting the ends of the hybridized probe with each other to form a circularized-probe, which interlocks with the target strand through catenation;

    c) cleaving the circularized probe such that the opened probe has the primer binding sequences oriented to enable polymerase chain reaction amplification of the probe identification sequence;

    d) hybridizing a probe detection oligonucleotide to the probe identification sequence present in the said probe, where the probe detection oligonucleotide is cleavably linked to a mass tag;

    e) performing a primer extension reaction by providing a primer capable of hybridizing to the primer binding sequence upstream of the probe identification sequence and extending said primer with a polymerase having 5′

    exonuclease activity, so as to cleave the mass tag from the probe detection oligonucleotide;

    f) detecting the mass tag by mass spectrometry; and

    g) detecting the presence of a target nucleic acid in the sample.

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