Compositions and methods for quantifying a nucleic acid sequence in a sample
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.
1. A method for detecting a plurality of distinct reaction products produced in the course of a single reaction, the method comprising:
(a) contacting a target nucleic acid molecule under substantially isothermal conditions with a polymerase, two or more primer oligonucleotides, each of which specifically binds to a complementary sequence on the target nucleic acid molecule, a nicking enzyme, and a detectable polynucleotide probe, wherein each of the primer oligonucleotides comprises one or more 2′ modified nucleotides in the sequence complementary to the target nucleic acid molecule;
(b) generating amplicons comprising at least a portion of said target nucleic acid molecule; and
(c) detecting a signal specific for oligonucleotide probe hybridization to the target nucleic acid molecule or amplicon thereof, wherein the signal indicates the quantity of the target nucleic acid molecule or amplicon thereof.
2. The method of claim 1 , wherein step (c) is carried out in real time.
3. The method of claim 1 , wherein the 2′ modification is selected from the group consisting of 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-hydroxyl, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH 2 —O-2′-bridge, 4′-(CH 2 ) 2 —O-2′-bridge, 2′-LNA, and 2′-O—(N-methylcarbamate) and base analogs.
4. The method of claim 1 , wherein the one or more 2′ modified nucleotides are positioned at the 3′ end of the sequence complementary to the target nucleic acid molecule; and/or
wherein the one or more 2′ modified nucleotides are positioned at the 5′ end of the sequence complementary to the target nucleic acid molecule.
5. The method of claim 1 , wherein the probe is N,N-dimethyl-N-[4-[(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine (SYBR™ green) or a Molecular Beacon.
6. The method of claim 1 , wherein the probe is a non-amplifiable detectable polynucleotide probe comprising at least about 10 nucleotides that are complementary to a target sequence, a detectable moiety, and a polymerase-arresting molecule, wherein the polymerase arresting molecule prevents a polymerase from amplifying the probe under conditions that otherwise support polymerase activity.
7. The method of claim 1 , wherein two or more 2′ modified nucleotides are contiguous.
8. The method of claim 1 , wherein 5 contiguous 2′-O-methyl modified nucleotides are positioned at the 3′ end of the sequence complementary to the target nucleic acid molecule; and/or
wherein 5 contiguous 2′-O-methyl modified nucleotides are positioned at the 5′ end of the sequence complementary to the target nucleic acid molecule.
9. A method for detecting a plurality of distinct reaction products in a test sample, the method comprising:
(a) contacting a test sample comprising at least one target nucleic acid molecule under substantially isothermal conditions with one or more polymerases, two or more primer oligonucleotides, each of which specifically binds to a complementary sequence on the target nucleic acid molecule, one or more nicking enzymes, and a detectable polynucleotide probe, wherein each of the primer oligonucleotides comprises one or more 2′ modified nucleotides in the sequence complementary to the target nucleic acid molecule;
(b) generating amplicons comprising at least a portion of said target nucleic acid molecule; and
(c) detecting a signal specific for oligonucleotide probe hybridization to the at least one target nucleic acid molecule or amplicon thereof, wherein the signal indicates the quantity of the at least one target nucleic acid molecule present in the sample or amplicon thereof.
10. The method of claim 1 , wherein step (c) is carried out in real time.
11. The method of claim 1 , wherein the test sample comprises a pathogen.
12. The method of claim 11 , wherein the pathogen is a virus, bacteria, yeast or fungus.
13. The method of claim 9 , wherein the test sample is a biological sample or an environmental sample.
14. The method of claim 13 , wherein the biological sample is a biological fluid, cell, or tissue sample.
15. The method of claim 14 , wherein the biological fluid is selected from the group consisting of blood, serum, plasma, amniotic fluid, sputum, urine, semen, vaginal secretion, cerebrospinal fluid, lymph, tear fluid, feces, and gastric fluid.
16. The method of claim 9 , wherein the target nucleic acid molecule or amplicon thereof is detected in a lateral flow device.
17. The method of claim 9 , wherein the method is carried out at a point of care.
18. The method of claim 9 , wherein step (c) is carried out at the endpoint of the reaction.
19. A method of quantifying a plurality of distinct reaction products in a nicking and extension amplification reaction, the method comprising:
(a) contacting a target nucleic acid molecule under substantially isothermal conditions with one or more polymerases, two or more primer oligonucleotides, each of which specifically binds to a complementary sequence on the target nucleic acid molecule, one or more nicking enzymes, and a detectable polynucleotide probe, wherein each of the primer oligonucleotides comprises one or more 2′ modified nucleotides positioned in the sequence complementary to the target nucleic acid molecule;
(b) generating amplicons comprising at least a portion of said target nucleic acid molecule; and
(c) detecting a signal specific for oligonucleotide probe hybridization to the target nucleic acid molecule or amplicon thereof, wherein the signal indicates the quantity of the target nucleic acid molecule present in the sample or an amplicon thereof.
20. A method of amplifying a plurality of target nucleic acid molecules, the method comprising:
(a) contacting the plurality of target nucleic acid molecules under substantially isothermal conditions with one or more polymerases, two or more primer oligonucleotides, each of which specifically binds to a complementary sequence on a target nucleic acid molecule, and one or more nicking enzymes, wherein each of the primer oligonucleotides comprises one or more 2′ modified nucleotides positioned in the sequence complementary to said target nucleic acid molecule; and
(b) generating amplicons comprising at least a portion of said target nucleic acid molecules.
21. The method of claim 20 , further comprising detecting the amplicons with a detectable probe.
22. The method of claim 20 , wherein the one or more 2′ modified nucleotides are positioned at the 3′ end of the sequence complementary to said target nucleic acid molecule; and/or
wherein the one or more 2′ modified nucleotides are positioned at the 5′ end of the sequence complementary to said target nucleic acid molecule.
23. The method of claim 22 , wherein one or more 2′ modified nucleotides positioned at the 5′ end of the sequence complementary to said target nucleic acid molecule are separated from a nick site by 1, 2, 3, 4, 5 or more unmodified nucleotides.
24. The method of claim 20 , wherein two or more 2′ modified nucleotides are contiguous.
25. The method of claim 20 , wherein the plurality of target nucleic acid molecules is derived from a test sample.
26. A kit for detecting a plurality of distinct reaction products produced in the course of a single reaction, the kit comprising one or more polymerases, one or more nicking enzymes, two or more primer oligonucleotides, each of which specifically binds to a complementary sequence on a target nucleic acid molecule and comprises one or more 2′ modified nucleotides positioned at the 3′ end of the sequence complementary to said target nucleic acid molecule, and directions for use in detecting a plurality of distinct reaction products produced in the course of a single reaction.