XÂY DỰNG QUY TRÌNH CHẨN ĐOÁN BIẾN THỂ ĐA HÌNH ĐƠN NUCLEOTIT RS1800629 TRÊN VÙNG KHỞI ĐỘNG CỦA GEN TNF- BẰNG KỸ THUẬT GIẢI TRÌNH TỰ SANGER VÀ PCR-RFLP

Abstract

Introduction: A single nucleotide polymorphism (SNP) at promoter region of TNF-α gene, TNF-α-308G/A (rs1800629), which directly affects binding affinity of transcription factors to promoter region, leads to alter TNF-α gene expression and increase TNF-α level. In addition to the standard technique for SNPs detection, Sanger sequencing, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method is developed to determine rs1800629 with basic laboratory equipments, without requiring expensive chemicals and, additionally, provides highly reliable results. Determining rs1800629 at affordable cost is a favorable premise for researching on the effects of rs1800629 on many important pathogenetic and physiological characteristics. Objective: Establishing a molecular procedure to identify the TNF-α-308G/A (rs1800629) variant by Sanger sequencing and PCR-RFLP method. Method: Constructing Sanger sequencing procedure with self-designed primer pairs to determine a 400-500 bp DNA amplicon containing rs1800629. Cloning the control recombinant DNA plasmids containing respectively G allele and A allele of rs1800629 by using TA cloning method. Optimizing the conditions of PCR-RFLP protocol with NcoI restriction enzyme and applying rs1800629 detection procedure on 5 known genotype DNA samples of volunteers. Result: (1) Successfully constructed a Sanger sequencing process to detect rs1800629 variant using self-designed primer pairs; (2) created 2 DNA plasmids containing the G allele (wild-type) and A allele (variant) of the rs1800629 as genotype control samples, and (3) optimized the PCR-RFLP procedure to determine the rs1800629 variant including cleavage of PCR product with NcoI enzyme, and electrophoresis. All 5 DNA samples of volunteers with genotypes GG (3 samples), GA (1 sample) and AA (1 sample) were correctly diagnosed by PCR-RFLP method. Conclusion: The kit including Sanger sequencing, PCR-RFLP method and DNA plasmid controls was completed to determine TNF-α-308G/A (rs1800629) variant at promoter region of TNF-α gene.