Analysis of FOXO3a gene polymorphism associated with asthma

Abstract

Single-nucleotide polymorphisms (SNP) represent genetic variation in a human population; 99.9% of the DNA sequence is identical and remaining 0.1% of DNA contains sequence variants. Around ten million SNPs are identified post human genome project. Identification of SNPs associated with disease phenotype has diagnostic potential and may predict drug response. Several technologies genotype thousands of SNPs simultaneously, for instance, genotyping arrays, gene-chip arrays, bead array technology, pyrosequencing, TaqMan approach, etc., however Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) remains a simplest, reliable, and cost effective method. In PCR-RFLP, a fragment that is to be analyzed for a mutation is amplified from genomic DNA using PCR, digested using appropriate restriction enzyme and then separated by gel electrophoresis. Sets of primer are used to amplify a fragment of interest and are designed in such a way that it flanks the polymorphic site and is located in such a way as to generate uneven sized fragments upon restriction endonuclease cleavage of the amplified PCR products. FOXO3a plays a key role in maintaining immunoregulation and is associated with several inflammatory diseases. Here, we report the PCR-RFLP protocol developed for detection of polymorphism in FOXO3a gene (rs13217795) by amplifying a 667 bp fragment followed by restriction endonuclease analysis using PagI to obtain RFLP patterns.