Estabilishment And Application Of Novel SNP Genotyping Method

This article mainly introduces the design and application of novel genotyping methods,which can be divided into three parts: The first part creates and applies allele-specific PCR method based on high-fidelity enzyme for SNP discrimination.The second part discusses the design and application of fluorescence probe melting curve genotyping method.The third part explores the use of UNG enzyme to solve the problem caused by primer dimer..Part 1:Allele-specific PCR is one of the most widely used and common SNP genotyping techniques.This method relies on the differential amplification efficiency of correctly and incorrectly paired base pairs by Taq(Thermus Aquaticus)polymerase in PCR reaction,and the subsequent enlargement of the differential signal in the following PCR reactions to achieve the ultimate goal of SNP genotyping.Therefore,the sensitivity of Taq polymerase to mismatched base pairs is one of the key factors for the implementation of this technology.In the past,to carry out allele-specific PCR,various methods such as introducing mutations into primers or using specially synthesized primers with modified bases were employed to indirectly enhance the specificity of Taq polymerase,the reason of which is the fact Taq polymerase tolerates mismatches during amplification reactions.To address this issue,this study introduced a novel high-fidelity Taq polymerase in allele-specific PCR,and expanded this strategy for multiple reaction while ensuring high specificity,and then the method was applied to the genotyping experiment of chromosomal replacement mice.In the multiple allele-specific PCR experiment,this study constructed a two-step amplification strategy combining capillary electrophoresis detection:at the first step,low-cycles number,low-primer concentration amplification using high-fidelity Taq polymerase was carried out,and then the reaction products were diluted and added to the second reaction system.At the second step,the signal was further amplified using universal sequences so that the fluorescent signal can be detected by capillary electrophoresis.To verify the feasibility of this strategy,three loci were selected from samples the mouse with partial segment replacement on chromosome 8 for specificity testing.After that,this strategy was used for primer concentration testing,optimization and adjustment of the second round system,and sensitivity detection.Finally,nine SNP genotypes of wild-type chromosome 1 replacement mice were used in the further test.The results showed that even with the normal primer design,Taq polymerase has good specificity and still can perform accurate genotyping.The minimum primer concentration can reach between 0.003 μ mol/L to 0.006 μ mol/L.The second round system verification mainly demonstrated that the configuration of the second round system has little effect on less SNP numbers genotyping.The detection limit of the strategy can reach below 0.078 ng/μL,which demonstrates high detection sensitivity.Ultimately,this strategy successfully conducted multiple SNP genotyping on chromosome 1 replacement mice.In conclusion,it is proved to be a suitable method for low-cost genotyping work with certain throughput and high accuracy requirements and provides a reference option for genotyping tasks of multiple loci.Part 2:The fluorescence probe melting curve method for genotyping is mainly based on the fluorescence changes of probes targeting sequence during the melting process.During melting,there is a distance change between the fluorescence group and quenching group of the probe in the extension state when binds the DNA single strand and in the free state when breaks off from the DNA single strand.Based on the different chain dynamics state produced by the base change on the target template,corresponding fluorescence melting peaks are generated to determine the SNP information on the target.In China,there are few reports on this technology compared to other genotyping methods,and its application has not been widely popularized yet,leaving much room for further development.To promote the development and application of this technology,this study established and discussed the technical process in detail,developed the application of related loci,summarized a series of issues regarding probe design,system configuration,detection systems,and finally formed a set of experimental schemes that can be repeated.Firstly,we used mutation primers to study the impact of probe on genotyping results,focusing on the significance of base distribution for SNP genotyping and using mutation primers to modify the sequence of P53 gene sites that are hard to genotype.We conducted SNP genotyping experiments on mice with mutated segments on chromosome 8 and studied the sensitivity of multi-probe melting curves and their performance in different testing systems.Then we selected ten SNP loci of medical research value for further experiments,including the study of probe concentration,primer concentration,and other parameters.Finally,we combined different loci to form a set of multiple melting curve genotyping system,which was used for genotyping testing of human non-extraction oral samples.Finally,we compared UNAfold simulation software with experimental melting curve graphs,summarizing key points for probe sequence design and ultimately completing the construction of the overall detection process.Part 3:The detection of primer dimers during amplification has hindered the detection of target products,especially in multiple amplifications where primer dimer amplification side reactions seriously affect target product synthesis.Therefore,this study explores the use of UNG enzyme to eliminate primer dimers and verifies and confirms the feasibility of the experimental scheme.In this experiment,primers containing d U bases were used in the first round of amplification.After adding universal primers and UNG enzymes,the second round of amplification was carried out to eliminate the effects of primer dimers on PCR detection.The problems such as insufficient amplification yield and imperfect amplification matching were optimized,and these problems were eliminated or mitigated by different measures.This experiment introduces a unique method for eliminating primer dimers,laying the foundation for further research in this field.