Recombinase Polymerase Amplification

Abstract

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. Further, the improved processivity of the disclosed methods may allow amplification of DNA up to hundreds of megabases in length.

1 .- 187 . (canceled) 
     
     
         188 . A reagent mixture for nucleic acid amplification comprising;
 T4 bacteriophage UvsX;   at least one DNA polymerase; and   a crowding agent.   
     
     
         189 . The reagent mixture of  claim 188 , wherein the crowding agent is selected from the group consisting of polyethylene glycols, dextran, Ficoll and a combination thereof. 
     
     
         190 . The reagent mixture of  claim 189 , wherein said polyethylene glycol is selected from the group consisting of PEG1450, PEG3000, PEG8000, PEG10000, PEG compound (molecular weight 15,000 to 20,000), and combinations thereof. 
     
     
         191 . The reagent mixture of  claim 188 , wherein the at least one polymerase is selected from the group consisting of prokaryotic polymerase, eukaryotic polymerase and phage-encoded polymerase. 
     
     
         192 . The reagent mixture of  claim 191 , wherein said eukaryotic polymerase is selected from the group consisting of pol-α, pol-β, pol-δ, and a combination thereof. 
     
     
         193 . The reagent mixture of  claim 191 , wherein the procaryotic polymerase is selected from the group consisting of  E. coli  DNA polymerase I Klenow fragment, bacteriophage T4 gp43 DNA polymerase,  B. stearothermophilus  polymerase (Bst),  B. subtilis  Phi-29 polymerase,  B. subtilis  polymerase I (Bsu),  E. coli  DNA polymerase I,  E. coli  DNA polymerase II,  E. coli  DNA polymerase III,  E. coli  DNA polymerase IV,  E. coli  DNA polymerase V and a combination thereof. 
     
     
         194 . The reagent mixture of  claim 188 , wherein the at least one polymerase includes at least one DNA polymerase which lacks 3′-5′ exonuclease activity. 
     
     
         195 . The reagent mixture of  claim 188 , wherein the at least one polymerase comprises a DNA polymerase with strand displacing properties. 
     
     
         196 . The reagent mixture of  claim 188 , further comprising a first nucleic acid primer and optionally a second nucleic acid primer. 
     
     
         197 . The reagent mixture of  claim 196 , wherein the reagent mixture comprises a first nucleic acid primer and a second nucleic acid primer. 
     
     
         198 . The reagent mixture of  claim 196 , wherein the first and second nucleic acid primers are selected from the group consisting of DNA, RNA, PNA, LNA, morpholino backbone nucleic acid, phosphorothiorate backbone nucleic acid, and a combination thereof. 
     
     
         199 . The reagent mixture of  claim 197 , further comprising a third nucleic acid primer. 
     
     
         200 . The reagent mixture of  claim 199 , further comprising a fourth nucleic acid primer. 
     
     
         201 . The reagent mixture of  claim 188 , wherein the T4 bacteriophage UvsX is temperature-sensitive. 
     
     
         202 . The reagent mixture of  claim 188 , further comprising at least one single stranded DNA binding protein. 
     
     
         203 . The reagent mixture of  claim 202 , wherein said single stranded DNA binding protein is selected from the group consisting of  E. coli  SSB, T4 gp32 and combinations thereof. 
     
     
         204 . The reagent mixture of  claim 188 , further comprising a buffer, ATP or an ATP analog, dNTPs or a mixture of dNTPs and ddNTPs. 
     
     
         205 . The reagent mixture of  claim 188 , further comprising a T4 bacteriophage uvsY. 
     
     
         206 . The reagent mixture of  claim 188 , wherein the dNTP(s) is/are selected from the group consisting of dATP, dGTP, dCTP, dTTP. 
     
     
         207 . The reagent mixture of  claim 188 , wherein the ATP or ATP analog is selected from ATP, ATP-γ-S, ATB-β-S, ddATP or a combination thereof. 
     
     
         208 . The reagent mixture of  claim 188 , further comprising a target nucleic acid sequence. 
     
     
         209 . The reagent mixture of  claim 196 , at least one primer comprises a 3′ blocking group. 
     
     
         210 . A composition comprising:
 the reagent mixture of  claim 188 ;   a target nucleic acid; and   at least on nucleic acid primer.   
     
     
         211 . A kit comprising:
 a T4 bacteriophage UvsX;   at least one DNA polymerase; and   a crowding agent.   
     
     
         212 . The kit of  claim 211 , further comprising a first nucleic acid primer and optionally a second nucleic acid primer. 
     
     
         213 . The kit of  claim 211 , further comprising a buffer, ATP or ATP analog, dNTPs, or a mixture of dNTPs and ddNTPs. 
     
     
         214 . The kit of  claim 212 , further comprising a third primer and optionally a fourth primer. 
     
     
         215 . The kit of  claim 211 , wherein the crowding agent is selected from the group consisting of polyethylene glycols, dextran, Ficoll and a combination thereof.