Manual

Product Number: RPA001

Shipping and Storage

Store at -20℃ to avoid repeated freezing and thawing.

Description

Recombinant enzyme polymerase amplification technology is a new technology that involves multiple enzymes and proteins to achieve nucleic acid index amplification under constant temperature conditions. It has the characteristics of sensitive reaction, high efficiency, and high cost-effectiveness. Recombinant enzyme polymerase amplification technology simulates DNA amplification in vivo and produces the target fragment under isothermal conditions. This technology mainly relies on the recombinase UvsX, single stranded binding protein gp32, and strand displacement DNA polymerase Bsu. The recombinase UvsX is capable of breaking double stranded DNA without heating. At the beginning of the recombinase polymerase amplification reaction, the recombinase UvsX binds to the primer with the participation of ATP, forming nucleic acid protein complexes that can scan for target double stranded DNA complementary to the primer sequence. Subsequently, the nucleic acid protein complex invades the 5 ‘end site to form a D-shaped loop. The single chain binding protein gp32 binds to the displaced single chain to stabilize it. At the same time, the recombinant enzyme uvsx leaves the 3 ‘end of the oligonucleotide and is degraded before being utilized by DNA polymerase. Afterwards, the strand displacement DNA polymerase Bsu binds to the free 3 ‘end of the nucleic acid protein complex for strand extension, forming a new complementary strand. During the process of chain extension, the newly synthesized single chain pairs with the original complementary chain. The above steps are repeated to achieve exponential growth of DNA.

Our company provides recombinant expression of UvsX protein with a molecular weight of~44kDa, high activity, and good stability. We constructed an efficient RPA isothermal amplification system using self-produced UvsX protein (as shown in the figure below).

A RPA isothermal amplification system was constructed using self-produced recombinant proteins such as Uxsx, UxsX, gp32, Bsu, etc.

M is a marker, and 1, 2, 3, and 4 are different RPA constant temperature amplification products.

Application

DNA isothermal amplification; RPA amplification; Genetic testing.

Specification

1. Dosage form: liquid or freeze-dried powder
2. Storage buffer: 50mM Tris HCl, 50mM KCl, 1mM DTT, 0.1mM EDTA, pH 8.0
3. Molecular weight: Approximately 44 KDa (detected by SDS-PAGE)
4. Purity: ≥ 90% (detected by SDS-PAGE)
5. Thermal deactivation: 60℃, 10 minutes

Quality control

1. Detection of residual nucleases: Incubate 500U of this enzyme with 0.6μg of λ – Hind III at 37℃ for 16 hours, and the electrophoresis band of DNA remains unchanged.
2. Detection of residual endonuclease: 500U of this enzyme and 0.6μg Supercoiled pBR322 DNA were incubated at 37℃ for 4 hours, and the electrophoresis band of DNA remained unchanged.
3. E. coli DNA residue detection: The residual nucleic acid in 50 U of this product was detected by TaqMan qPCR specific for E. coli 16s rDNA, and the E. coli genome residue was less than 10 copies.

Note

This reagent is only used for research and development or production, and is strictly prohibited from being used in human or animal experiments.