Quantitative Real Time PCR Assay Using FRET Dual-Labeled Primers
AssigneeLife Technologies Corporation
InventorsWayne Ge, Yue Ling Ng
Life of this patent
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Abstract
This specification generally relates to non-radioactive methods of non-radioactive real-time PCR using FRET dual-labeled primers.
Claims41
A method for the quantification or detection of one or more target nucleic acid molecules in a sample during nucleic acid synthesis, the method comprising:
- a) mixing one or more target nucleic acid molecules with one or more fluorescently-labeled oligonucleotides, wherein said one or more oligonucleotides are labeled with a fluorophore and a quencher and said oligonucleotide undergoes a detectable change in fluorescence upon extension of said one or more target nucleic acid molecules;
- b) incubating said mixture with a polymerase under conditions sufficient to synthesize one or more nucleic acid molecules complementary to all or a portion of said one or more target nucleic acid molecules, said one or more synthesized nucleic acid molecules comprising said one or more oligonucleotides;
- and
- c) detecting the presence or absence or quantifying the amount of said one or more synthesized nucleic acid molecules by measuring said fluorophore, wherein the extension is by at least 3 nucleotides.
A method for amplifying a double-stranded nucleic acid molecule, comprising:
- a) providing a first and second primer, wherein said first primer is complementary to a sequence within or at or near the 3′-terminus of the first strand of said nucleic acid molecule and said second primer is complementary to a sequence within or at or near the 3′-terminus of the second strand of said nucleic acid molecule;
- b) hybridizing said first primer to said first strand and said second primer to said second strand in the presence of one or more polymerases, under conditions such that said primers are extended to result in the synthesis of a third nucleic acid molecule complementary to all or a portion of said first strand and a fourth nucleic acid molecule complementary to all or a portion of said second strand;
- c) denaturing said first and third strands, and said second and fourth strands;
- and repeating the above steps one or more times, wherein one of said first and second primers is dual-labeled with a fluorophore and a quencher;
- and
- wherein said dual-labeled primer undergoes a detectable change in fluorescence upon extension of said one or more labeled primers to said nucleic acid molecule, wherein extension is by at least 3 nucleotides.
A fluorescently-labeled oligonucleotide comprising both a fluorophore and a quencher, wherein the fluorophore and the quencher are separated at a distance such that when the oligonucleotide is bound to a target nucleic acid and extended by at least three nucleotides the fluorophore is not quenched by the quencher, and when the oligonucleotide is not bound to a target nucleic acid the fluorophore is quenched by the quencher.
A kit for the quantification or detection of one or more target nucleic acid molecules in a sample during nucleic acid synthesis, comprising:
- (a) a polymerase, and
- (b) a dual-labeled oligonucleotide comprising a fluorophore and a quencher, wherein the quencher and fluorophore are separated at a distance such that when the dual-labeled oligonucleotide bound to the nucleic acid molecule is not extended the fluorophore is quenched by the quencher and when the dual-labeled oligonucleotide bound to the nucleic acid molecule is extended the fluorophore is not quenched by the quencher.
A composition comprising:
- (a) a polymerase, and
- (b) a dual-labeled oligonucleotide comprising a fluorophore and a quencher, wherein the quencher and fluorophore are separated at a distance such that when the dual-labeled oligonucleotide bound to the nucleic acid molecule is not extended the fluorophore is quenched by the quencher and when the dual-labeled oligonucleotide bound to the nucleic acid molecule is extended the fluorophore is not quenched by the quencher.
Dependencies are inferred from the claim text.