Investigation Of Magnetic Enrichment Multiplex Polymerase Chain Reaction Amplification And Application

Because of the advantages of strong specificity, high sensitivity,rapidness, convenience, and so on, the polymerase chain reaction （PCR）has been an important researching method in the field of life sciences.Enhancing the specificity and improving the sensitivity is the focus ofPCR technology researching.In this work, magnetic γ-Fe₂O₃nanoparticles with average size of30nm were prepared. The nanoparticles functionalized with streptavidinwere proved to be bio-active and could be used in biology test.The magnetic nanoparticles were combined with multiplex PCRtechnology to perform magnetic enrichment multiplex PCR amplificationto enhance the specificity and sensitivity of PCR technology.First, magnetic enrichment single PCR amplification was performedin order to analyze the feasibility of this method. In this experimentprocess, first, the target sequence was enriched at the surface of magneticnanoparticles functionalized with streptavidin after hybridization with thebiotin-modified specific primer. Then single target sequence was obtainedby denaturation, and PCR amplification was performed with the primersof purpose sequence. After optimizing the experiment conditions, it canbe concluded that the optimal hybridization temperature between specificprimers and target sequence is52.5°C, and the dosage of magneticnanoparticles γ-Fe₂O₃functionalized with streptavidin is90μg, and thelowest concentration of target sequence can be detected as low as5×10-10ng/μL. This method is feasible.The primers of M235T site and A-6G site of AGT gene and A1298Csite and C677T site of MTHFR gene were designed. With this four pairsof primers, magnetic enrichment multiplex PCR amplification wasperformed on the base of magnetic enrichment single PCR amplification.Through the optimization experiment, it can be concluded that theannealing temperature of magnetic enrichment multiplex PCRamplification is56°C, the dosage of25mmol/L of MgCl2is2.0μL,10 μmol/L of dNTP is1.5μL,5U/μL of Taq enzyme is0.7μL,10mmol/Lof C677T primers is1μL,10mmol/L of M235T primers is0.7μL,10mmol/L of A1298C primers is1μL,10mmol/L of A-6G primers is1.5μL in30μL amplification system. Simultaneously, the sensitivity ofmagnetic enrichment multiplex PCR amplification obtained is5×10-9ng/μL.Combining the technology of universal primer PCR amplificationand gene chip, a simple application of magnetic enrichment multiplexPCR amplification was performed for the SNP genotyping of the foursites.