Recombinase polymerase amplification
View Patent ↗This disclosure describes related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of recombinase and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods have the advantage of not requiring thermocycling or thermophilic enzymes, thus offering easy and affordable implementation and portability relative to other amplification methods. Further disclosed are conditions to enable real-time monitoring of RPA reactions, methods to regulate RPA reactions using light and otherwise, methods to determine the nature of amplified species without a need for gel electrophoresis, methods to improve and optimize signal to noise ratios in RPA reactions, methods to optimize oligonucleotide primer function, methods to control carry-over contamination, and methods to employ sequence-specific third ‘specificity’ probes. Further described are novel properties and approaches for use of probes monitored by light in dynamic recombination environments.
1. A method for identifying a polymorphism in a target nucleic acid, the method comprising:
a) producing an amplicon using a mixture comprising:
1) a target nucleic acid comprising a first strand and a second strand;
2) a recombinase;
3) a polymerase;
4) at least one nucleic acid primer; and
5) a crowding agent; and
b) identifying a polymorphism in said target nucleic acid by detecting said polymorphism in said amplicon.
2. The method of claim 1 wherein detecting said polymorphism in said amplicon comprises contacting said amplicon with a nucleic acid probe.
3. The method of claim 2 wherein said nucleic acid probe comprises a detectable label.
4. The method of claim 2 wherein said nucleic acid probe is an immobilized nucleic acid probe.
5. The method of claim 4 wherein an array of probes comprises said immobilized nucleic acid probe.
6. The method of claim 1 wherein said mixture comprises a first nucleic acid primer and a second nucleic acid primer; and the recombinase is in contact with the first nucleic acid primer to form a first nucleoprotein primer and the recombinase is in contact with the second nucleic acid primer to form a second nucleoprotein primer.
7. The method of claim 6 wherein producing an amplicon comprises:
(a) contacting the first and second nucleoprotein primers to the target nucleic acid thereby forming a first double stranded structure at a first portion of the first strand and forming a second double stranded structure at a second portion of the second strand such that the 3′ ends of the first nucleic acid primer and the second nucleic acid primer are oriented toward each other on the same target nucleic acid molecule;
(b) extending the 3′ end of the first and second nucleoprotein primers with the polymerase and dNTPs to generate a first and second double stranded nucleic acid and a first and second displaced strand of nucleic acid; and
(c) repeating the contacting and extending steps until a desired degree of amplification is reached.
8. The method of claim 1 wherein said amplicon is double stranded.
9. The method of claim 1 wherein said amplicon is single stranded.
10. The method of claim 1 wherein detecting said polymorphism in said amplicon comprises sequencing said amplicon.
11. The method of claim 2 wherein said nucleic acid probe comprises a nucleotide sequence complementary to a polymorphism in said amplicon.
12. The method of claim 2 wherein said nucleic acid probe hybridizes said amplicon at a region of said amplicon comprising a polymorphism.
13. The method of claim 1 wherein said mixture further comprises a helicase that disrupts imperfect nucleic acid hybrids.
14. The method of claim 13 wherein said helicase is PriA helicase, DnaB helicase, dda helicase, gp41 helicase, or RuvAB helicase.
15. The method of claim 1 wherein said mixture further comprises a nuclease that disrupts imperfect nucleic acid hybrids.
16. The method of claim 1 wherein said polymorphism is a tandem repeat.
17. The method of claim 1 wherein said polymorphism is a single nucleotide polymorphism.
18. The method of claim 1 wherein said polymorphism is a deletion, an insertion, an inversion, a frameshift mutation, or a crossover.
19. The method of claim 1 wherein the amplicon comprises a label.
20. The method of claim 6 wherein said first nucleic acid primer comprises a detectable label and/or said second nucleic acid primer comprises a detectable label.
21. The method of claim 6 wherein said first nucleic acid primer comprises a nucleotide sequence that is complementary to a polymorphism and/or said second nucleic acid primer comprises a nucleotide sequence that is complementary to a polymorphism.
22. A system for identifying a polymorphism in a target nucleic acid, the system comprising a composition comprising:
1) at least one recombinase;
2) at least one polymerase;
3) a target nucleic acid;
4) a crowding agent; and
5) a first nucleic acid primer and optionally a second nucleic acid primer.
23. The system of claim 22 further comprising an array of probes.
24. The system of claim 22 further comprising a labeled nucleic acid probe.
25. The system of claim 22 further comprising a nucleic acid sequencing apparatus.
26. The system of claim 22 wherein said first nucleic acid primer comprises a detectable label.