Compositions for 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 . - 72 . (canceled) 
     
     
         73 . A method of identifying the presence or absence of a target nucleic acid associated with a specific genotype, comprising:
 contacting a sample with reagents to perform recombinase polymerase amplification (RPA), the reagents comprising:   a first primer comprising a unique 3′-sequence complementary to the target nucleic acid; and   a second primer;   performing RPA to amplify the target nucleic acid;   analysing the product of the RPA, and;   identifying the presence of the genotype of interest based on the concentration or amount of amplified target nucleic acid.   
     
     
         74 . The method of  claim 73 , wherein the genotype is associated with a species pathogenic to a mammal, or to a plant. 
     
     
         75 . The method of  claim 74  wherein the mammal is a human. 
     
     
         76 . The method of  claim 74  wherein the mammal is selected from the group consisting of bovine, equine, porcine, canine, feline. 
     
     
         77 . The method of  claim 74  wherein the plant is a food crop plant. 
     
     
         78 . The method of  claim 74  wherein the plant is a non-food crop plant. 
     
     
         79 . The method of  claim 77 , wherein the food crop plant is selected from the group consisting of wheat, barley, oats, rye, maize, potatoes, tomatoes, rice, bananas, apples and pears. 
     
     
         80 . The method of  claim 78 , wherein the non-food crop plant is selected from the group consisting of tobacco, bamboo, willow and reed. 
     
     
         81 . The method of  claim 74 , wherein the pathogen is selected from the group consisting of a virus, a  bacterium , a fungus and a parasite. 
     
     
         82 . The method of  claim 81 , wherein the virus is selected from the group consisting of influenza, rubella, varicella-zoster, hepatitis A, hepatitis B, other hepatitis viruses, herpes simplex, polio, smallpox, human immunodeficiency virus, vaccinia, rabies, Epstein Barr, retroviruses, and rhinoviruses. 
     
     
         83 . The method of  claim 81 , wherein the bacteria is selected from the group consisting of  Escherichia coli, Mycobacterium tuberculosis, Salmonella  sp.,  Chlamydia  sp. and  Streptococcus  sp. 
     
     
         84 . The method of  claim 81 , wherein the parasite is selected from the group consisting of  Plasmodium  sp.,  Trypanosoma  sp.,  Toxoplasma gondii , and  Onchocerca  sp. 
     
     
         85 . The method of  claim 73 , wherein the first primer is complementary to a first nucleic acid associated with a first genotype but not to a second nucleic acid associated with a second genotype by at least one base at its 3′ end. 
     
     
         86 . The method of  claim 85 , wherein the first primer is not complementary to the second nucleic acid associated with the second genotype by at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases at its 3′ end. 
     
     
         87 . The method of  claim 85 , wherein the first primer is completely non-complementary to the second nucleic acid associated with the second genotype. 
     
     
         88 . The method of  claim 73 , wherein analysing the product of the RPA comprises determining the size or sequence of the amplified nucleic acid. 
     
     
         89 . The method of  claim 88 , wherein determining the size of amplified nucleic acid comprises a technique selected from the group consisting of agarose gels, PAGE gels, mass spectroscopy, pulsed field gels, gene chips, sucrose sedimentation. 
     
     
         90 . The method of  claim 88 , wherein determining the sequence of the amplified nucleic acid comprises a technique selected from the group consisting of Sanger sequencing, Maxam-Gilber sequencing, pyro-sequencing, sequencing by hybridisation (SBH). 
     
     
         91 . The method of  claim 85 , wherein the first genotype is a genetic disease genotype and the second genotype is a normal genotype. 
     
     
         92 . The method of  claim 85 , wherein the first genotype is a normal genotype and the second genotype is a genetic disease genotype. 
     
     
         93 . The method of  claim 73 , wherein the genotype is a point mutation, a deletion, an insertion, an inversion, a frameshift mutation, a crossover event, or the presence or absence of multiple copies of genetic sequences. 
     
     
         94 . The method of  claim 73 , wherein the genotype is associated with a disease or predisposition to a disease. 
     
     
         95 . The method of  claim 73 , wherein the second primer comprises a unique 3′-sequence complementary to the target nucleic acid.