Construction Of Recombinase-aid Nucleic Acid Amplification System And Its Using In SNP Genotyping

Nucleic Acid in vitro Amplification (NAA) used for exponential growth frommicroscale DNA template is a high-efficient nucleic acid amplification technology, whichis widely applied to molecular biology, medical science, medicolegal expertise, etc. Basedon the principle of NAA technology, RAA (Recombinase Aid Amplification) is developedas an in vitro isothermal amplification, employed the cooperative amplification model thatRecombinase invade DNA double strand, Single Strand Binding protein (SSB) combinessDNA and DNA Polymerase amplify, rather than temperature cycling model to unwindingDNA double strand which is known in PCR. RAA system is construction of recombnaseUvsX, crowding agent protein UvsY, single strand binding protein Gp32and polymeraseKlenow (exo-) at the core. In order to detect the advantagement and defect between PCRsystem and constructed RAA system, this experiment is comparation in aspects ofcensitivity, specifity, fidelity and template interference. In order to enhance the specifity ofRAA system and applied RAA system to SNP detection, RAA system is optimized insolution pH and salt concentration.From the comparation result, RAA system performedwell than PCR system in system’s censitivity, specific, fidelity. The template interference issimilar in both PCR and RAA. The result of SNP detection in RAA system is highlyconsistent with cequencing.