Method for detecting nucleic acid amplification using self-quenching fluorescence probe

  • US 5,538,848 A
  • Filed: 11/16/1994
  • Issued: 07/23/1996
  • Est. Priority Date: 11/16/1994
  • Status: Expired due to Term
First Claim
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1. A method for monitoring nucleic acid amplification comprising:

  • performing nucleic acid amplification on a target polynucleotide using a nucleic acid polymerase having 5'"'"'-3'"'"' nuclease activity, a primer capable of hybridizing to said target polynucleotide, and an oligonucleotide probe capable of hybridizing to said target polynucleotide, 3'"'"' relative to said primer,said oligonucleotide probe having a fluorescent reporter molecule and a quencher molecule capable of quenching the fluorescence of said reporter molecule,said oligonucleotide probe existing in at least one single-stranded conformation when unhybridized where said quencher molecule quenches the fluorescence of said reporter molecule, said oligonucleotide probe existing in at least one conformation when hybridized to said target polynucleotide where the fluorescence of said reporter molecule is unquenched, the fluorescence intensity of said reporter molecule being greater than the fluorescence intensity of said quencher molecule when said probe is hybridized to said target;

    polynucleotide,said nucleic acid polymerase digesting said oligonucleotide probe during amplification to separate said reporter molecule from said quencher molecule; and

    monitoring the fluorescence of said reporter molecule, the generation of fluorescence corresponding to the occurrence of nucleic acid amplification.

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