Journal
CHEMBIOCHEM
Volume 15, Issue 15, Pages 2268-2274Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/cbic.201402250
Keywords
isothermal amplification; nucleotide analogues; oligonucleotides; polymerase chain reaction; recombinase polymerases
Funding
- DARPA ADEPT [HR0011-11-2-0018]
- Defense Threat Reduction Agency [HDTRA1-13-1-0004]
- Nucleic Acids Licensing LLC
- Firebird Biomolecular Sciences LLC
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Recombinase polymerase amplification (RPA) is an isothermal method to amplify nucleic acid sequences without the temperature cycling that classical PCR uses. Instead of using heat to denature the DNA duplex, RPA uses recombination enzymes to swap single-stranded primers into the duplex DNA product; these are then extended using a strand-displacing polymerase to complete the cycle. Because RPA runs at low temperatures, it never forces the system to recreate base-pairs following Watson-Crick rules, and therefore it produces undesired products that impede the amplification of the desired product, complicating downstream analysis. Herein, we show that most of these undesired side products can be avoided if the primers contain components of a self-avoiding molecular recognition system (SAMRS). Given the precision that is necessary in the recombination systems for them to function biologically, it is surprising that they accept SAMRS. SAMRS-RPA is expected to be a powerful tool within the range of amplification techniques available to scientists.
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