Methods and devices for measuring duplex unwinding or strand exchange efficiency
Provided herein are quantitative assays to evaluate duplex unwinding efficiency or strand exchange efficiency during isothermal amplification.
1 . A method for detecting unwinding or strand exchange of a double stranded nucleic acid, the method comprising:
providing a sample comprising a pre-annealed duplex, wherein the pre-annealed duplex comprises a target nucleic acid and a synthetic nucleic acid comprising an optional first detectable label; a primer comprising a second detectable label; an amplifying enzyme for amplifying nucleic acids; and an unwinding enzyme for unwinding or strand exchange double stranded nucleic acids;
incubating the sample under an isothermal amplification condition and for a time sufficient for nucleic acid amplification; and
detecting the second detectable label and, optionally, the first detectable label, if present,
wherein a ratio of the primer to the pre-annealed duplex is from 1:1 to 50:1.
2 . The method of claim 1 , wherein the synthetic nucleic acid comprises an initial primer, and wherein the initial primer comprises the first detectable label.
3 . The method of claim 2 , wherein said incubating further comprises:
extending the initial primer, thereby providing an extension product comprising the first detectable label;
unwinding the extension product;
annealing the primer comprising the second detectable label to a portion of the target nucleic acid; and
extending the primer, thereby providing a further extension product comprising the second detectable label.
4 . The method of claim 1 , wherein the synthetic nucleic acid comprises an extension product of an initial primer, and wherein the initial primer comprises the first detectable label.
5 . The method of claim 1 , wherein the synthetic nucleic acid comprises an elongated nucleic acid comprising about 20 or more nucleotides.
6 . The method of claim 5 , wherein the elongated nucleic acid comprises the first detectable label.
7 . The method of claim 1 , wherein said incubating further comprises:
unwinding the duplex;
annealing the primer comprising the second detectable label to a portion of the target nucleic acid; and
extending the primer, thereby providing an extension product comprising the second detectable label.
8 . The method of claim 1 , further comprising, prior to said providing:
annealing the target nucleic acid and the synthetic nucleic acid, thereby forming the pre-annealed duplex; and
optionally cooling the pre-annealed duplex.
9 . The method of claim 1 , further comprising, prior to said providing:
annealing the target nucleic acid and the synthetic nucleic acid, wherein the synthetic nucleic acid is configured to be an initial primer; and
extending the initial primer under a polymerase primer extension condition, thereby forming the pre-annealed duplex.
10 . The method of claim 1 , further comprising, after said incubating:
inactivating the sample using thermal inactivation and/or chemical inactivation.
11 . The method of claim 1 , further comprising prior to said detecting:
purifying the sample to remove one or more chemical or biological components.
12 . The method of claim 1 , wherein the synthetic nucleic acid and the primer comprises the same nucleic acid sequence.
13 . The method of claim 1 , wherein the synthetic nucleic acid is shorter than or the same length as the primer.
14 . The method of claim 1 , wherein the synthetic nucleic acid is longer than the primer.
15 . The method of claim 1 , wherein the synthetic nucleic acid and the primer has, independently, a length of about 5 to 100 nucleotides.
16 . The method of claim 1 , wherein the pre-annealed duplex comprises a 3′-tailed end and/or a 5′-tailed end.
17 . The method of claim 1 , wherein the pre-annealed duplex comprises at least one blunt end.
18 . The method of claim 1 , wherein the pre-annealed duplex has a length of about 15 to 500 base pairs.
19 . The method of claim 1 , wherein the first and second detectable labels are same or different.
20 . The method of claim 19 , wherein the first and second detectable labels are, independently, provided at a 5′-end or internally.
21 . The method of claim 19 , wherein the synthetic nucleic acid comprises a plurality of first detectable labels, and/or wherein the primer comprises a plurality of second detectable labels.
22 . The method of claim 19 , wherein the first and second detectable labels are selected from the group consisting of a fluorescent label, a radioactive label, a chemiluminescent label, or a dye.
23 . The method of claim 1 , wherein the sample further comprises 3′-amino-2′,3′-dideoxyribonucleotide 5′-triphosphates (nNTPs), a divalent ion, a denaturant, a buffer, and/or a salt.
24 . The method of claim 1 , wherein the amplifying enzyme and/or the unwinding enzyme is selected from the group consisting of a helicase, a recombinase, a polymerase, a reverse transcriptase, a thermophilic form thereof, a thermostable form thereof, and a recombinant form thereof.
25 . The methods of claim 1 , wherein the isothermal amplification condition comprises a temperature of about 20 to 75° C.
26 . The method of claim 1 , wherein said detecting comprises electrophoresis analysis, optionally on a microfluidic device.
27 . The method of claim 26 , wherein the electrophoresis analysis comprises analysis of fragment size of an amplicon comprising the second detectable label.
28 . The method of claim 27 , wherein the electrophoresis analysis further comprises comparing the fragment size of the amplicon to a labeled size standard.