Isothermal amplification components and processes
The technology relates in part to methods and compositions for isothermal amplification of nucleic acids.
1. A method for detecting a target nucleic acid sequence in a sample, the method comprising:
(a) contacting a sample RNA in a sample with a reverse transcriptase to generate a cDNA;
(b) contacting the cDNA with an enzyme having a hyperthermophile polymerase activity to generate a double-stranded DNA (dsDNA), wherein the dsDNA comprises a target nucleic acid sequence, and wherein the target nucleic acid sequence comprises a first strand and a second strand complementary to each other;
(c) amplifying the target nucleic acid sequence under an isothermal amplification condition, wherein the amplifying comprises contacting the dsDNA with:
(i) a first primer and a second primer, wherein the first primer is capable of hybridizing to a sequence of the first strand of the target nucleic acid sequence, and the second primer is capable to hybridizing to a sequence of the second strand of the target nucleic acid sequence; and
(ii) the enzyme having a hyperthermophile polymerase activity, thereby generating a nucleic acid amplification product, wherein the nucleic acid amplification product comprises:
(1) the sequence of the first primer, and the reverse complement thereof,
(2) the sequence of the second primer, and the reverse complement thereof, and
(3) a spacer sequence flanked by (1) the sequence of the first primer and the reverse complement thereof and (2) the sequence of the second primer and the reverse complement thereof, wherein the spacer sequence is 1 to 10 bases long; and
(d) detecting the nucleic acid amplification product, wherein the detecting is performed in 20 minutes or less from the time the dsDNA is contacted with (c)(i) the first and second primers and (c)(ii) the enzyme having a hyperthermophile polymerase activity,
wherein the method does not comprise using any enzymes other than the reverse transcriptase and the enzyme having a hyperthermophile polymerase activity.
2. The method of claim 1 , wherein step (c) further comprises determining the amount of the dsDNA that comprises the target nucleic acid sequence in the sample.
3. The method of claim 1 , wherein the sample RNA is a cellular RNA, a mRNA, a microRNA, a bacterial RNA, or a viral RNA.
4. The method of claim 1 , wherein the target nucleic acid sequence is a bacterial nucleic acid sequence or a viral nucleic acid sequence.
5. The method of claim 1 , wherein the enzyme having a hyperthermophile polymerase activity has an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:8 or a functional fragment thereof.
6. The method of claim 1 , wherein the enzyme having a hyperthermophile polymerase activity has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:8.
7. The method of claim 1 , wherein the enzyme having a hyperthermophile polymerase activity is a polymerase comprising the amino acid sequence of SEQ ID NO: 8.
8. The method of claim 1 , wherein the enzyme having a hyperthermophile polymerase activity has low or no exonuclease activity.
9. The method of claim 1 , wherein the sample comprises RNA from prokaryotes or eukaryotes.
10. The method of claim 9 , wherein the sample comprises RNA from a virus or a bacterium.
11. The method of claim 1 , wherein the method does not comprise contacting the dsDNA with a single-stranded DNA binding protein prior to or during step (c).
12. The method of claim 1 , wherein amplifying the target nucleic acid sequence is performed at a constant temperature of about 55 degrees Celsius to about 75 degrees Celsius.
13. The method of claim 12 , wherein amplifying the target nucleic acid sequence is performed at a constant temperature of about 65 degrees Celsius.
14. The method of claim 1 , wherein the first primer, the second primer, or both is about 8 to 16 bases long.
15. The method of claim 1 , wherein the nucleic acid amplification product is about 20 to 40 bases long.
16. The method of claim 1 , wherein the spacer sequence comprises a portion of the target nucleic acid sequence.
17. The method of claim 16 , wherein the spacer sequence is 1 to 5 bases long.
18. The method of claim 1 , further comprising contacting the nucleic acid amplification product with a signal-generating oligonucleotide capable of hybridizing to the amplification product, wherein the single-generating oligonucleotide comprises a fluorophore, a quencher, or both.
19. The method of claim 1 , wherein detecting the nucleic acid amplification product comprises detecting a fluorescent signal.
20. The method of claim 19 , wherein the fluorescent signal is from a molecular beacon.
21. The method of claim 1 , wherein the method is performed in a single reaction vessel.
22. The method of claim 1 , wherein the sample RNA is contacted with the reverse transcriptase and the enzyme having a hyperthermophile polymerase activity simultaneously.
23. The method of claim 22 , wherein the sample RNA is contacted with the reverse transcriptase, the enzyme having a hyperthermophile polymerase activity, and the first and second primers simultaneously.
24. The method of claim 1 , wherein the first primer, the second primer, or both comprises one or more of DNA bases, modified DNA bases, or a combination thereof.