Establishment Of High Resolution Melting Curve Analysis Technology (HRM) System And Its Application In Gene Polymorphism And Mutation Detection

Since being developed in2003, the High-Resolution Melting Curve Analysis (HRM or HRMA) technology has been quickly applied to various fields of studies because of its obvious advantages such as faster and high sensitivity. HRM can detect DNA sequence variations in real time through changes in the melting temperature of a DNA duplex, and it only needs a pair of primers detect the bases changes of alleles rather than target site specificity primers or probe. High sensitivity, specificity, simple, fast and efficient associated with high-throughput, close-tube operation and low cost when compared with traditional methods that require electrophoresis and techniques using labeled probes, make HRM a suitable cost-effective approach. In this study, we first established a fast and efficient HRM detection system through detecting the ALDH2polymorphisms, and then detected the birth defects related gene MTHFR and MTRR gene polymorphism loci by HRM, proving that HRM could be applied to clinical single-gene disorders detection fitfully with high accuracy and speed. At the same time, we also tried to detect the efficiency of gene mutations cutting with the transcription activator-like effectoer nucleases (TALEN) by HRM, and the results show that HRM could be useful for detecting the mutation efficiency of unknown mutation points. In summary, HRM is a prior method which is fast, effective, economic and facilitate to the detection of single gene disorders and mutations.Part Ⅰ Establish aldehyde dehydrogenase (ALDH2) gene polymorphism detection system by HRM.Purpose:This part of study aims to explore influence of the DNA concentration and purity on the sensitivity and accuracy of HRM, and to build polymorphic loci HRM detection system. Method:Collecting96plasma samples of healthy persons. The DNA was extracted from blood and quantitied. Design the specific HRM primers and detect rs671of ALDH2gene though HRM and direct sequencing, compare both the test results. Result:By HRM, we detected67ADLH2*1/1(G/G) wild type,26ADLH2*1/2(G/A) heterozygous and three ADLH2*2/2(A/A) missense mutations type of96cases, the results are consistent with the sequencing results. Conclusion:HRM is a simple, fast, economical, efficient and high-throughput genetic analysis method, and it can be used for the rapid detection of ALDH2gene mutation, and can be applied to the detection of other gene polymorphisms.Part II Detection of birth defects related to subgenotype methylene-tetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) gene polymorphism by HRMPurpose:This part of study aims to dicuss the accuracy of the HRM for SNP detection by detecting two SNP loci of MTHFR and a SNP locus of MTRR, and then provide reference for the rapid detection of birth defects related gene polymorphisms and treatment clinical neonatal. Method:Collecting96plasma samples of healthy persons. The DNA was extracted from blood and quantitied. Design the specific HRM primers and detect c.677C> T and c.1298A> C of MTHFR gene and c.66A> G of MTRR gene though HRM and direct sequencing, compare both the test results. Result:By HRM, we detected61cases of MTHFR c.677C> T type,31C/T type, and4T/T type of96cases, and50cases of MTHFR c.1298A> C type,30A/C type,16C/C type, and61cases of MTRR c.66A> G type,51A/A type,40A/G type, and5G/G type, the results are consistent with the sequencing results. Conclusion:HRM can quickly and accurately detect birth defects related gene polymorphism, and it can be applied as a highly effective clinical detection method.Part Ⅲ Using HRM to detect the efficiency of gene mutations cutting by TALENPurpose:This part of study aims to investigate the feasibility of detecting gene mutation by HRM and to establish a rapid, accurate and efficient gene mutation model detection platform. Method:Construct specific knockout TALEN plasmids of the MSTN gene, and then conduct cell transfection experiments, the tail vein of mice fast red experiments and mouse zygotes microinjection experiments to gain the gene knockout DNA, and then design specific primers for HRM detection, detect and and sequence all samples and compare their results. Result:By HRM, DNA mutation typing results are seven holes293T cells DNA sample typing three groups which clearly separated from the control group, and in the experiment group there are different variation of different typing.10cases of mouse liver DNA samples were parted into three groups, No.1as a group. No.2and3as a group, and the remaining controls were divided into one group.6out of45mice survived in the fertilized egg after microinjection get mutation, choose No.4and6as a parent, and mate them with normal mice to produce F1generation, detect F1and choose some of them to produce F2generation. The whole process of fast detecting is conduct by HRM, compared with the sequencing results, the correct rate of HRM is100%. Conclusion: HRM is a preferred method for rapid detection of mutations with its high accuracy, and it is applicable in the diversity of the screening saving a lot of time and cost.