Recombinase Polymerase Amplification (RPA) enzymes
Recombinase Polymerase Amplification (RPA) system is fast (10-20 min) and suitable for both DNA and RNA analytes. RPA rely on the biological properties of the bacterial RecA recombinase protein, or its prokaryotic and eukaryotic homologues. RPA process employs three enzymes: a recombinase, a single-stranded (ss) DNA-binding protein (SSB) and a strand-displacing polymerase. At first, recombinase coats on ssDNA (primers) to form nucleoprotein filaments. These filaments can then scan targeted double stranded DNA for sequences complementary to those of coated primers. Then, the nucleoprotein filaments initiate a 5’-strand invasion at the site of homology forming a structure known as D-loop. The strand invasion is stabilized by single strand binding protein. After that, strand extension takes place at the free 3’-end of the nucleoprotein filaments by a strand displacing DNA polymerase to synthesize a new complementary strand. During strand extension, the new synthesized strand displaces the originally paired strand. With the use of two opposing primers, exponential amplification of the target sequence with RPA can be achieved at a constant temperature (25-42ºC).
G-biosciences offers a selection of enzymes related to RPA system. All RPA enzymes are manufactured under ISO certified facility.
- T4 UvsX DNA recombinase mediates DNA strand exchange between homologous chromosomes. The protein forms a right-handed nucleoprotein complex on single ssDNA called the presynaptic filament that can search for homology in duplex DNA and pair the recombining DNA molecules to form a DNA joint molecule.
- T4 UvsY recombination mediator protein plays important roles in homologous recombination (HR). During HR, the UvsX recombinase competes with the prebound single-stranded binding protein gp32 for DNA-binding sites and UvsY stimulates this filament nucleation event.
- T4 GP32 is a single-stranded DNA binding protein with key role in DNA replication, recombination and repair. T4 gp32 binds cooperatively to single stranded DNA and stimulates cell-free DNA synthesis directed by the T4-encoded DNA polymerase.
- Bsu DNA Polymerase, Large fragment is from Bacillus subtilis and is modified to retain its 5’→ 3’ DNA polymerase activity with its 5’→ 3’ exonuclease domain removed. It also lacks the 3’→ 5’ exonuclease activity.