Recombinase polymerase amplification

Abstract

This disclosure describe three related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of the bacterial RecA and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods has the advantage of not requiring thermocycling or thermophilic enzymes. Further, the improved processivity of the disclosed methods allow amplification of DNA up to hundreds of megabases in length.

1 . A RPA process of DNA amplification of a double stranded target sequence, said target sequence comprising a first and a second strand of DNA, comprising the steps of:
 (a) contacting a recombinase agent with a first and a second nucleic acid primer to form a first and a second nucleoprotein primer;   (b) contacting the first and second nucleoprotein primers to said double stranded target sequence thereby forming a first double stranded structure at a first portion of said first strand and form a double stranded structure at a second portion of said second strand such that the 3′ ends of said first nucleic acid primer and said second nucleic acid primer are oriented toward each other on the same template nucleic acid molecule;   (c) extending the 3′ end of said first and second nucleoprotein primer with one or more polymerases and dNTPs to generate a first and second double stranded nucleic acid and a first and second displaced strand of nucleic acid   (d) continuing the reaction through repetition of (b) and (c) until a desired degree of amplification is reached.   
     
     
         2 . The method of  claim 1 , wherein in step (c), the displaced strand of nucleic acid is amplified by a concurrent RPA reaction. 
     
     
         3 . The method of  claim 2  wherein the displaced strand of nucleic acid is amplified by:
 (a) contacting the first or the second nucleoprotein primer to said displaced strand; and 
 (b) extending said first or second nucleoprotein primer with a polymerase and dNTPs. 
 
     
     
         4 . The process of  claim 1  wherein said first and said second displaced strand is at least partially complementary to each other and can hybridize to form a daughter double stranded nucleic acid which can serve as double stranded template nucleic acid in a subsequent step (b) and step (c). 
     
     
         5 - 7 . (canceled) 
     
     
         8 . The process of  claim 1  wherein at least one primer is partially single stranded and partially double stranded, wherein said primer is comprised of a longer invading-strand which possesses a specific 3′ primer sequence and a shorter non-invading strand, which is complementary to the 5′ end of the longer invading strand. 
     
     
         9 . The process of  claim 8  wherein said first nucleic acid primer comprise a first non invading strand and said second nucleic acid primer comprise a second non-invading strand and said first and second non-invading strand are complementary to each other. 
     
     
         10 . The process of  claim 9  wherein said invading strand, said non-invading strand, or both said invading strand and said non-invading strand are labeled with a detectable moiety. 
     
     
         11 - 15 . (canceled) 
     
     
         16 . The process of  claim 1  wherein step (d) causes the displacement of the non-invading strand. 
     
     
         17 . The process of  claim 9  wherein the invading strand and the non-invading strand are labeled and the separation of the invading strand and the non-invading strand is detected. 
     
     
         18 . A process according to  claims 1  in which the released strand of one partly double-stranded invading oligonucleotide is the complementary sequence to the released strand of the other double-stranded invading oligonucleotide. 
     
     
         19 . A process according to  claims 18  in which the released oligonucleotide is complementary to, and hybridizes to a single-stranded target template and prime DNA synthesis from its 3′ end. 
     
     
         20 - 22 . (canceled) 
     
     
         23 . A process according to  claim 1  in which the recombinase agent is temperature-sensitive, wherein said temperature sensitive recombinase agent possessing strand-invasion activity at a permissive temperature and no strand-invasion activity at a non permissive temperature. 
     
     
         24 - 30 . (canceled) 
     
     
         31 . The RPA process of  claim 1  wherein said first portion and second portion spans an intervening target DNA sequence. 
     
     
         32 - 48 . (canceled) 
     
     
         49 . The RPA process of  claim 1  wherein said process is performed without temperature induced melting of the template nucleic acid. 
     
     
         50 - 59 . (canceled) 
     
     
         60 . A process of nested RPA amplification comprising the steps of:
 (a) amplifying a region of DNA using the RPA process of  claim 1  using a first and a second primer to produce a first amplified product;   (b) amplifying said amplified product using a third and a fourth primer using the RPA process of  claim 1  to produce a second amplified product, wherein said second amplified product is a smaller sequence contained within said first amplified product.   
     
     
         61 . The process of  claim 60  wherein said first primer, said second primer, said third primer and said fourth primer are each different. 
     
     
         62 . The process of  claim 60  wherein said one of said first and said second primer is identical to one of said third and fourth primer. 
     
     
         63 . A RPA process of DNA amplification of a double stranded target molecule comprising the steps of
 (a) contacting a recombinase agent with a first and a second nucleic acid primer to form a first and a second nucleoprotein primer;   (b) contacting the first and second nucleoprotein primers to said double stranded target sequence thereby forming a first double stranded structure at a first portion of said target molecule and a second double stranded structure at a second portion of said target molecule such that the 3′ ends of said first nucleic acid primer and said second nucleic acid primer are oriented toward each other on the same template nucleic acid molecule;   c) contacting said target sequence to primosome assembly proteins and a clamp loader/polymerase III holoenzyme complex to form a replication fork at said first portion of target molecule and a second replication fork at said second portion of the target molecule;   d) contacting said target molecule to a DNA polymerase III core, a DNA polymerase I, a primase, a helicase, and a ligase to allow coupled leading and lagging strand DNA synthesis and the migration of the replication forks towards each other;   continuing the reaction through repetition of steps (b), (c) and (d) until a desired degree of amplification is reached.   
     
     
         64 . (canceled) 
     
     
         65 . The method of  claim 63 , which is performed in the presence of single strand DNA binding protein. 
     
     
         66 - 67 . (canceled) 
     
     
         68 . The method of  claim 63  wherein the clamp loader is a DnaX clamp loader complex. 
     
     
         69 - 72 . (canceled)