QUY TRÌNH GIẢI TRÌNH TỰ SANGER MỘT SỐ BIẾN THỂ ĐA HÌNH ĐƠN NUCLEOTIDE TRÊN CÁC GEN PNPLA3, TM6SF2, MBOAT7 VÀ GCKR LIÊN QUAN ĐẾN BỆNH GAN NHIỄM MỠ KHÔNG DO RƯỢU

Abstract

Objective: Develop Sanger sequencing and investigate five variants on PNPLA3, TM6SF2, MBOAT7 and GCKR genes related to non-alcoholic fatty liver disease (NAFLD). Materials and methods: Optimized the DNA extraction process with erythrocyte lysis by ACK solution, designed 5 pairs of primers specific for variants, optimized the pairing temperature of the PCR reaction and optimized Sanger sequencing reaction on an Applied Biosystems 3500 Series Genetic Analyzer from Thermo Fishser. Apply the entire optimized Sanger sequencing process to 4 blood samples of volunteers to evaluate the specifications and characteristics of the variants. Results: Successfully built Sanger sequencing process including: optimization of erythrocyte volume and lysis time with ACK to obtain DNA of standard purity, designing 5 pairs of primers effectively amplifying the five variants of interest, optimizing the concentration of DNA participating in the sequencing reaction. Obtained 4/4 participants with at least one of the 5 variants of interest with all sequencing results having a QVB > 90%. Conclusion: Established and optimized Sanger sequencing to identify single nucleotide polymorphisms. Initially applying the process on volunteers, as a basis for surveys with representative sample sizes to approach and manage NAFLD in molecular perpestive in Vietnam.