TITLE:
Tetra Primer ARMS PCR Optimization to Detect Single Nucleotide Polymorphism of the KLF14 Gene
AUTHORS:
Ana Sofia Zabala, Miriam Ester Vasquez Gomez, Micaela Fernanda Álvarez, Susana Siewert
KEYWORDS:
rs972283 (A/G), KLF14 Gene, Type 2 Diabetes Mellitus
JOURNAL NAME:
Open Access Library Journal,
Vol.4 No.12,
December
5,
2017
ABSTRACT: GWAS (genome-wide association studies) have associated Type 2 Diabetes Mellitus with the single nucleotide polymorphism (SNPs) rs972283 (A/G) of KLF14 gene with in a global population. The most common techniques used to analyze SNPs are time consuming, multi-step process and require expensive instruments. Therefore, in order to overcome these problems, we have developed a new, rapid and cost effective T-ARMS PCR assay to genotype rs972283. However, the optimization step can be hardworking and laborious. Hence, we propose to demonstrate and discuss critical steps for its development, in a way to provide useful information. In a first step, we design and validate two specific primer pair for T-ARMS PCR. Later, the amplification conditions were optimized for DNA concentration, annealing temperature, Taq DNA polymerase units and primers concentration. The last one was considered the main interference factor for a correct amplification and appropriate band intensity. Finally, the results obtained by T-ARMS PCR were concordant with sequencing. T-ARMS PCR assay developed in our laboratory for genotyping rs972283 (A/G) of KLF14 gene is time saving and cost-effective compared to the available methods used for SNP studies.