Development Of Alzheimer's Disease Gene Diagnosis Technology

Alzheimer's disease(AD)is a neurodegenerative disease with a strong genetic predisposition.Single nucleotide polymorphism(SNP)refers to the polymorphism of DNA sequence caused by the mutation of single nucleotide in the genome of chromosomes.Many phenotypic differences and susceptibility to drugs or diseases are associated with SNP.The purpose of this project is to screen a stable and reliable isothermal amplification technology,and design a kit for the diagnosis of alzheimer's disease by detecting the SNP of AD related genes,so as to carry out early diagnosis and prevention of this disease.Through the literature research and preliminary experimental results,we preliminary screening 65 SNPs loci associated with AD,and mainly explored based on ring Mediated Isothermal Amplification techniques(Loop-Mediated Isothermal Amplification,LAMP)of three kinds of improved method:1.Smart amplificaton prcoess(SMAP).This method uses asymmetric primer design and the TaqMuts mismatch repair protein to inhibit non-specific amplification and reduce the occurrence of false positive.In this paper,we first extract human glioma cell line(U87)genomic DNA,and designs the identification of ALDH2 rs671 primer group to verify the feasibility of SMAP method,compared with the classification results agree with generation sequencing results,and the data display TaqMuts mismatch repair proteins can obviously decrease nonspecific amplification effect,just 15 min can be observed that the classification results,and 40 min can complete the whole process of amplification.However,subsequent genotyping of APOE rs429358 was not successful.2.Based on the ring connection reaction Mediated Isothermal Amplification techniques(Ligation-Loop-Mediated Isothermal Amplification,L-LAMP)through the design of two complementary hairpin probe and the target sequences,the Taq DNA ligase two probe can be formed under the action of dumbbell shape structure,the structure can be used as the intermediate LAMP reaction just start the LAMP reaction.In this paper,we designed two hair pin probes to detect rs429358,but the results were not stable.After analysis,it was found that the mismatching of hairpin probes or the formation of dimers may lead to the failure of the connection reaction or the decrease of the efficiency,thus affecting the stability and accuracy of LAMP reaction.3 Modified-Loop-Mediated Isothermal Amplification(M-LAMP)primer design simple,the method for SNP detection efficiency and accuracy are improved significantly.In this paper,we designed two sets of primers for detecting the gene PICALM rs3851179 and the gene APOE rs429358.The data showed that the amplification results were consistent with the sequencing results.In addition to the complementary part of the target sequence,the 5 'end of primer FIP and BIP is connected with a complementary palindrome sequence.The complementary palindrome sequence can be designed as a general sequence according to specific practical requirements,and the 3' end is the SNP site to be tested.Different from the traditional LAMP product accumulation,the products formed by the special structure of the 5 'end of primers FIP and BIP can act as primers for the next round of amplification.In this way,the amplification products are in series with each other,and eventually a large number of target sequences are formed.Conclusion: SMAP method and L-LAMP method have some limitations in SNP detection and cannot be widely used.On the contrary,the M-LAMP method has high specificity and stability,and has obvious advantages in primer design,long fragment amplification and SNP detection.As a new nucleic acid amplification technology or SNP detection technology,M-LAMP method has a good application prospect.