Recombinase polymerase amplification
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 recombinase polymerase amplification (RPA) method of detecting target DNA in a biological sample, comprising;
(a) contacting the biological sample with a lytic agent to form a lysed sample;
(b) forming a mixture comprising components of a recombinase polymerase amplification (RPA) reaction and a portion of the lysed sample, wherein the components of the RPA reaction comprise a recombinase, a polymerase, a crowding agent, a first nucleic acid primer, and a second nucleic acid primer;
(c) maintaining the mixture under conditions to amplify a target nucleic acid in the sample;
(d) determining the presence or amount of target nucleic acid, thereby detecting the presence or absence of a target nucleic acid.
2. The method of claim 1 , wherein the lysed sample is added to the mixture without separation or purification of the target nucleic acid from the lysed sample.
3. The method of claim 1 , wherein the biological sample is blood.
4. The method of claim 1 , wherein the target nucleic acid is genomic DNA.
5. The method of claim 1 wherein the recombinase is selected from UvsX, RecA, and combinations thereof.
6. The method of claim 1 , wherein the polymerase includes at least one DNA polymerase which lacks 3′-5′ exonuclease activity.
7. The method of claim 1 , wherein the polymerase comprises a DNA polymerase with strand displacing properties.
8. The method of claim 1 , wherein the crowding agent is present at a concentration between 1 to 12% of a weight or a volume of the mixture.
9. The method of claim 1 , wherein the crowding agent is selected from polyethylene glycols, dextran, ficoll and a combination thereof.
10. The method of claim 9 wherein the crowding agent comprises polyethylene glycol at a concentration between 1 to 12% of a weight or a volume of the mixture.
11. The method of claim 10 , wherein said polyethylene glycol is selected from PEG1450, PEG3000, PEG8000, PEG10000, PEG compound (molecular weight 15,000 to 20,000), and combinations thereof.
12. The method of claim 1 , wherein the first and second nucleic acid primers are selected from DNA, RNA, PNA, LNA, morpholino backbone nucleic acid, phosphorothiorate backbone nucleic acid, and a combination thereof.
13. The method of claim 1 wherein the mixture further comprises at least one recombinase loading protein.
14. The method of claim 13 , wherein the recombinase loading protein is selected from T4 uvsY, E. coli recO, E. coli recR and combinations thereof.
15. The method of claim 1 wherein the recombinase is temperature-sensitive.
16. The method of claim 1 , the mixture further comprising at least one single stranded DNA binding protein.
17. The method of claim 16 , wherein said single stranded DNA binding protein is E. coli SSB, and/or T4 gp32.
18. The method of claim 1 , wherein the first nucleic acid primer and/or the second nucleic acid primer comprises a 3′ blocking group.
19. The method of claim 1 , the mixture further comprising trehalose.