Compositions and Methods for Quantifying a Nucleic Acid Sequence in a Sample
AssigneeEnvirologix Inc.
InventorStephen A. Judice
Life of this patent
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Abstract
The present invention features compositions and methods for quantifying detection of a target oligonucleotide in a sample in real time. These methods are compatible with target oligonucleotides amplified using a NEAR reaction.
Claims34
A non-amplifiable detectable polynucleotide probe, the probe comprising at least about 10 nucleotides that are complementary to a target sequence, a detectable moiety, and a polymerase-arresting molecule.
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6-7. (canceled)
10-11. (canceled)
15-16. (canceled)
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24-29. (canceled)
A method for monitoring in real time a target nucleic acid molecule in a NEAR reaction, which comprises
- (a) contacting a target nucleic acid molecule under substantially isothermal conditions with a polymerase, two primer/template oligonucleotides, each of which specifically binds to a complementary sequence on the target nucleotide molecule, a nicking enzyme, a heteroduplex specific nicking enzyme, and a detectable oligonucleotide probe;
- (b) generating amplicons comprising a target sequence that binds the detectable oligonucleotide probe;
- and
- (c) detecting a signal in real time, thereby quantitating the target nucleic acid molecule.
A method for monitoring in real time a target nucleic acid molecule in a test sample, the method comprising:
- (a) contacting a target nucleic acid molecule under substantially isothermal conditions with a polymerase, two primer/template oligonucleotides, each of which specifically binds to a complementary sequence on the target nucleotide molecule, a nicking enzyme, a repair enzyme or proof reading enzyme, and a detectable oligonucleotide probe;
- (b) generating amplicons comprising a target sequence that binds the detectable oligonucleotide probe;
- and
- (c) detecting a signal in real time, thereby quantitating the target nucleic acid molecule.
32-38. (canceled)
A detectable circularizing oligonucleotide probe, wherein a portion of the 5′ end of the oligonucleotide probe is complementary to a portion of the 5′ end of the target nucleic acid molecule and comprises a fluorescent moiety;
- the 3′ end of the oligonucleotide probe is complementary to a portion of the 3′ end of the target nucleic acid molecule and comprises a quencher moiety;
- the complementary sequences at the 5′ and 3′ ends of the molecule are separated by a linker sequence;
- and the probe circularizes to bring its 5′ and 3′ ends into apposition when bound to a target nucleic acid molecule, thereby providing for emission of a detectable signal.
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A detectable oligonucleotide probe for quantitating a target nucleic acid molecule in a nicking and extension amplification reaction comprising
- (i) a hair pin shaped nucleic acid molecule comprising a double-stranded stem region and a single-stranded loop region, wherein at least a portion of said loop region is complementary to a target sequence;
- (ii) a polymerase arresting moiety at the 3′ end of the molecule, and
- (iii) a detectable moiety.
Dependencies are inferred from the claim text.