The Establishment Of System With Snapshot Technique In SCN5A Gene And Its Application In Sudden Cardic Death(SCD)

Objective:1?To develop a SNaPshot kit based on SCN5 A gene related to Sudden Cardic Death(SCD);2?To detect the type of SCD susceptibility mutation in SCN5 A gene in Shanxi population using the SNaPshot kit.Methods:The principle of this method was combined with multiplex PCR and SNaPshot technique to analysis of the mutations in ion channel gene:(1)The selected SNPs depended on the reported literature and NCBI database;(2)The singleplex PCR performed in each pair of primers to validate the specificity of each amplify product(A total of 30 pairs of primers).There was one SNP at least and five SNPs at most in these amplify produces;(3)To maximize the efficiency of amplification,divided 30 pairs of primers into two multiplex PCR system;(4)To detect the SNPs in two groups of multiplex PCR products,respectively(1st system contains 22 SNPs,2nd system contains 23 SNPs).The principle of this method combined SNaPshot tencnique and capillary electrophoresis(CE)to detect the type and position of mutations,and thus confirmed all the type of 45 SNPs in SCN5 A gene.According to the results of SNaPshot,confirmed all the SNPs with polymorphism by direct sequencing.Results:1.Established a SNaPshot system with 45 SNPs in SCN5 A gene successfully.2.According to the result of SNaPshot with these 45 SNPs,44 SNPs are in accordance with the recorded in NCBI database.The polymorphism was existed at rs1805124(H558R).3.Statically analysis performed according to the result in three groups of samples at rs1805124(H558R).The consequence of direct sequencing and the established system in this study are the same at rs1805124(H558R).Conclusion:The established SNaPshot kit aimed at SCN5 A gene,which related to sudden cardiac death(SCD),combined with multiplex PCR and SNaPshot technique to screen the polymorphism SNPs.(1)The SNaPshot kit in this study is a genetical detection tool to determine the cause of sudden cardiac death in forensic cases;(2)The SNaPshot kit in this study could be used to analysis the genetic background of the mutation with SCD and conclude the characteristic of family member;(3)The SNaPshot kit in this study provides the genetic evidence for birth and treanment.