Study On Multiple SNP Typing Scheme Based On AS-PCR Principle And Capillary Electrophoresis

Single Nucleotide Polymorphism(SNP)is a special genetic marker,which is of great significance for human medical research and forensic identification.With the in-depth study of genome-wide sequence information,there is a certain demand for SNP typing technology.The traditional detection methods include: first-generation sequencing technology,ligase detection technology,PCR-RFLP technology.Although these detection methods have high accuracy,problems such as fewer single detection sites,cumbersome steps,and time-consuming and laborious problems have seriously hindered their large-scale promotion.With the development of genetic testing technology,Next-Generation Sequencing(NGS),digital PCR technology and real-time PCR-based PCR platform(dye detection,probe method and high-resolution melting curve method)have emerged.These methods have different advantages,but their solution development process is complex and costly,making these detection methods difficult to comprehensively promote.This study developed a novel genotyping program to address the problems faced by current genotyping techniques.This study uses hairpin primers and universal fluorescent primers for typing,allowing rapid,accurate,and low-cost detection of samples.The advantages of this protocol are as follows:(1)Use hairpin primers to increase specificity,resulting in as few primer dimers as possible and non-specific amplification.In this study,hairpin primers with special structure were designed based on the principle of Allele Specific PCR(AS-PCR).It can effectively solve the problem that the primer dimer is generated due to the interaction between the primer and the primer,resulting in low amplification efficiency;and it can also effectively reduce the mismatch between the primer and the template,resulting in non-specific amplification.(2)Using the principle of primer competition to improve accuracy.Design a hairpin primer for each of the two genotypes at the same locus.When amplifying,the competition between the two primers was used to improve the accuracy.(3)Combining the use of universal fluorescent primers,single-time typing of multiple sites can be achieved.In this study,we used a typing scheme established by hairpin primers and universal fluorescent primers to detect genes involved in folate metabolism(C677T and A1298 C of MTHFR gene,A66 G of MTRR gene).The hairpin primer comprises,in turn,a hairpin sequence,a universal fluorescent primer binding sequence,and a specific sequence from the 5’ to the 3’ end.The so-called hairpin is such that the hairpin sequence at the 5’ end is complementary to the specific sequence at the 3’ end by about 10-13 bases,and the resulting structure is hairpin.During amplification,for specific templates,the hairpin primer will open to the target sequence of the template for stable amplification.Conversely,for non-specific templates,hairpin primers do not open,effectively inhibiting the production of non-specific products.The universal fluorescent primer binding sequence in the middle of the designed hairpin primer is identical to the sequence of the fluorescent group labeled universal fluorescent primer.When the hairpin primer is combined with the template for several cycles to produce a certain amount of product,the universal fluorescent primer will be combined with the product for extensive amplification such that the final product is labeled with a fluorescent group.Detection of the length and genotype can be performed on a capillary electrophoresis platform.In summary,this experiment uses a hairpin primer in combination with a universal fluorescent primer to genotype a site associated with folate metabolism.This protocol is a genotyping program with short time consumption,low price,high specificity and single-tube multi-site detection.