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.
123 . A recombinase polymerase amplification (RPA) composition comprising a recombinase, a polymerase, adenosine triphosphate, a low-activity oligonucleotide primer, and a high-activity oligonucleotide primer.
124 . The RPA composition of claim 123 , wherein said low-activity oligonucleotide primer has a length of less than approximately 30 nucleotides.
125 . The RPA composition of claim 123 , wherein said high-activity oligonucleotide primer has a length of approximately 30 or more nucleotides.
126 . The RPA composition of claim 123 , wherein said low-activity oligonucleotide primer has a length of 25-26 nucleotides.
127 . The RPA composition of claim 123 , wherein said low-activity oligonucleotide primer comprises a homopolymeric guanosine sequence at a 5′ end.
128 . The RPA composition of claim 123 , wherein said high-activity oligonucleotide primer comprises a homopolymeric cytosine sequence at a 5′ end.
129 . The RPA composition of claim 123 , wherein said low-activity oligonucleotide comprises a modified nucleotide base or modified nucleic acid backbone at a 3′ end.
130 . The RPA composition of claim 123 , wherein the concentration of said low-activity oligonucleotide primer is greater than the concentration of said high-activity oligonucleotide primer.
131 . The RPA composition of claim 123 , further comprising a crowding agent, a single-stranded DNA binding protein, and/or a reverse transcriptase.
132 . A recombinase polymerase amplification (RPA) composition comprising a recombinase, a polymerase, adenosine triphosphate, a pair of low-activity oligonucleotide primers, and a pair of high-activity oligonucleotide primers.
133 . The RPA composition of claim 132 , wherein said pair of low-activity oligonucleotide primers comprises a first oligonucleotide primer having a length of less than approximately 30 nucleotides and a second oligonucleotide primer having a length of less than approximately 30 nucleotides.
134 . The RPA composition of claim 132 , wherein said pair of high-activity oligonucleotide primers comprises a first oligonucleotide primer having a length of approximately 30 or more nucleotides and a second oligonucleotide primer having a length of approximately 30 or more nucleotides.
135 . The RPA composition of claim 132 , wherein said pair of low-activity oligonucleotide primers comprises a first oligonucleotide primer having a length of 25-26 nucleotides and a second oligonucleotide primer having a length of 25-26 nucleotides.
136 . The RPA composition of claim 132 , wherein said pair of low-activity oligonucleotide primers comprises a first oligonucleotide primer comprising a homopolymeric guanosine sequence at a 5′ end and a second oligonucleotide primer comprising a homopolymeric guanosine sequence at a 5′ end.
137 . The RPA composition of claim 132 , wherein said pair of high-activity oligonucleotide primers comprises a first oligonucleotide primer comprising a homopolymeric cytosine sequence at a 5′ end and a second oligonucleotide primer comprising a homopolymeric cytosine sequence at a 5′ end.
138 . The RPA composition of claim 132 , wherein said pair of low-activity oligonucleotide primers comprises a first oligonucleotide primer comprising a modified nucleotide base or nucleic acid backbone at a 3′ end and a second oligonucleotide primer comprising a modified nucleotide base or nucleic acid backbone at a 3′ end.
139 . The RPA composition of claim 132 , wherein the concentration of said pair of low-activity oligonucleotide primers is greater than the concentration of said pair of high-activity oligonucleotide primers.
140 . The RPA composition of claim 132 , further comprising a crowding agent, a single-stranded DNA binding protein, and/or a reverse transcriptase.
141 . A single-tube nesting RPA reaction mixture comprising a recombinase, a polymerase, adenosine triphosphate, a pair of low-activity oligonucleotide primers, a pair of high-activity oligonucleotide primers, and a target nucleic acid.
142 . The single-tube nesting RPA reaction mixture of claim 141 , wherein said pair of high-activity oligonucleotide primers is hybridized to the target nucleic acid and said pair of low-activity oligonucleotide primers is hybridized to an amplicon produced from said target nucleic acid.