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) amplifying a target nucleic acid sequence in a sample under an isothermal amplification condition, wherein the target nucleic acid sequence comprises a first strand and a second strand complementary to each other, and wherein the amplifying comprises contacting a double-stranded nucleic acid comprising the target nucleic acid sequence 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) an 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 or a portion thereof, or the reverse complement thereof,
(2) the sequence of the second primer or a portion thereof, or the reverse complement thereof, and
(3) a spacer sequence flanked by (1) and (2), wherein the spacer sequence is 1 to 10 bases long; and
(b) detecting the nucleic acid amplification product, wherein the detecting is performed in 20 minutes or less from the time the double-stranded nucleic acid is contacted with (a)(i) the first and second primers and (a)(ii) the enzyme having a hyperthermophile polymerase activity,
wherein the method does not comprise using any enzymes that are not a polymerase.
2. The method of claim 1 , wherein the amplifying under the isothermal amplification condition does not comprise using any enzymes other than the enzyme having a hyperthermophile polymerase activity.
3. The method of claim 1 , wherein the double-stranded nucleic acid is a genomic nucleic acid, a plasmid nucleic acid, a mitochondrial nucleic acid, a cellular nucleic acid, or an extracellular nucleic acid.
4. The method of claim 1 , wherein the double-stranded nucleic acid is a bacterial nucleic acid or a viral nucleic acid.
5. The method of claim 1 , further comprising before the amplifying step (a), generating the double-stranded nucleic acid by a reverse transcription reaction from a sample RNA using a reverse transcriptase.
6. The method of claim 5 , wherein the sample RNA is a cellular RNA, a mRNA, a microRNA, a bacterial RNA, or a viral RNA.
7. The method of claim 5 , wherein the method does not comprise using any enzymes other than the reverse transcriptase and the enzyme having a hyperthermophile polymerase activity.
8. The method of claim 5 , comprising contacting the sample RNA with the reverse transcriptase and the enzyme having a hyperthermophile polymerase activity simultaneously.
9. The method of claim 5 , comprising contacting the sample RNA with the reverse transcriptase, the enzyme having a hyperthermophile polymerase activity, the first primer, and the second primer simultaneously.
10. The method of claim 1 , further comprising determining the amount of the double-stranded nucleic acid that comprises the target nucleic acid sequence in the sample.
11. The method of claim 1 , wherein the target nucleic acid sequence is a bacterial nucleic acid sequence or a viral nucleic acid sequence.
12. 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.
13. 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.
14. The method of claim 1 , wherein the enzyme having a hyperthermophile polymerase activity has low or no exonuclease activity.
15. The method of claim 1 , wherein the sample is a specimen obtained or derived from a human, a non-human animal, a plant, a bacterium, a fungus, a virus, a protist, or a mixture thereof.
16. The method of claim 1 , wherein the sample is a bodily fluid sample, a tissue sample, or a mixture thereof from a human subject.
17. The method of claim 1 , wherein the method does not comprise contacting the double-stranded nucleic acid with a single-stranded DNA binding protein prior to or during step (a).
18. 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.
19. The method of claim 18 , wherein amplifying the target nucleic acid sequence is performed at a constant temperature of about 65 degrees Celsius.
20. The method of claim 1 , wherein the first primer, the second primer, or both is about 8 to 16 bases long.
21. The method of claim 1 , wherein the nucleic acid amplification product is about 20 to 40 bases long.
22. The method of claim 1 , wherein the spacer sequence comprises a portion of the target nucleic acid sequence.
23. The method of claim 22 , wherein the spacer sequence is 1 to 5 bases long.
24. The method of claim 1 , further comprising contacting the nucleic acid amplification product with an oligonucleotide probe capable of hybridizing to the amplification product.
25. The method of claim 24 , wherein the oligonucleotide probe comprises a fluorophore, a quencher, or both.
26. The method of claim 25 , wherein detecting the nucleic acid amplification product comprises detecting a fluorescent signal.
27. The method of claim 26 , wherein the fluorescent signal is from a molecular beacon.
28. The method of claim 1 , wherein the amplifying is performed under an isothermal amplification condition.
29. The method of claim 1 , wherein the method is performed in a single reaction vessel.
30. The method of claim 1 , wherein the first primer, the second primer, or both comprises one or more of DNA bases, one or more modified DNA bases, or any combination thereof.