Mutagenic primer-based PCR-RFLP assay for genotyping IRGM gene promoter variant rs4958843 (C/T)
Ambika Sharma1, Harish Changotra1
1Department of Biotechnology and Bioinformatics, Jaypee University of Information Technology, Solan, Himachal Pradesh, India.
Background:
Single-nucleotide polymorphisms play an important role in the susceptibility of many diseases, evolutionary studies, and genetic mapping. The rs4958843 in IRGM promoter is associated with tuberculosis and Crohn's disease. As this SNP is not present in any of the restriction sites, PCR-RFLP is not possible. Therefore, we have developed artificial-RFLP method to genotype this SNP.
Methods:
We designed forward primer with mismatches that resulted in the creation of a restriction site for enzyme NheI in the amplicon. Control samples of known genotypes were obtained by sequencing. The amplified product for SNP rs4958843 was digested with NheI restriction enzyme and resolved on an agarose gel to know the genotypes of the samples.
Results:
Results of sequencing and A-RFLP were concordant. The developed method was applied to genotype this polymorphism in 100 samples from healthy individuals. The allelic frequencies of SNP rs4958843 were C (0.16) and T (0.84), while corresponding genotypic distribution was CC (2), CT (29), and TT (69).
Conclusion:
The newly developed method is simple, easy, and cost-effective which could be used to genotype IRGM polymorphism -1161 C/T (rs4958843) in various populations in the replication studies and has its applicability in the clinical settings. The developed method was applied for genotyping samples from healthy individuals from North India. For the first time, we report the frequency of this polymorphism from this region.
