Modified Tetra-Primer ARMS PCR optimization for detection of ADIPOQ rs267729 SNP (C>G) in type 2 diabetes patients in San Luis, Argentina population

Reina Agustina Leonela Orozco; Sandra Beatriz Diaz; Maria Estela Soledad Baigorria; Susana Elfrida Siewert; Gomez Miriam Ester Vasquez

doi:10.30574/gscbps.2020.10.2.0030

Authors

Reina Agustina Leonela Orozco Laboratory of Diabetes, Department of Biochemistry and Biological Sciences, School of Chemistry, Biochemistry and Pharmacy, National University of San Luis, San Luis, Argentina.
Sandra Beatriz Diaz San Luis Hospital, San Luis, Argentina.
Maria Estela Soledad Baigorria San Luis Hospital, San Luis, Argentina.
Susana Elfrida Siewert Laboratory of Diabetes, Department of Biochemistry and Biological Sciences, School of Chemistry, Biochemistry and Pharmacy, National University of San Luis, San Luis, Argentina.
Gomez Miriam Ester Vasquez Laboratory of Diabetes, Department of Biochemistry and Biological Sciences, School of Chemistry, Biochemistry and Pharmacy, National University of San Luis, San Luis, Argentina.

DOI:

https://doi.org/10.30574/gscbps.2020.10.2.0030

Keywords:

Adipocytes, Inflammatory response, Insulin-Resistant, Genotyping

Abstract

The human Adiponectin (ADIPOQ) is an important adipocytokine that is secreted by adipocytes and plays a key role in the inflammatory response that is associated with insulin-resistant states and T2DM. The transversion of C to G (rs267729) in the promoter region of ADIPOQ gene could affect its transcription and secretion. Development of a new Modified Tetra Primer Amplification Refractory Mutation System - Polymerase Chain Reaction (MTPA-PCR) for detection of rs267729 (C>G) in the human promoter of ADIPOQ gene was sought. A MTPA-PCR for rs267729 polymorphism in a single-step PCR was carried out, and the results were confirmed by ASO-PCR technique in 25 type 2 diabetes mellitus patients and 25 controls. We have developed a sensitive single tube MTPA-PCR assay to detect rs267729 (C>G) in the human promoter of ADIPOQ gene, which allowed us to distinguish homozygous and heterozygous forms of this polymorphism successfully. Full accordance between ASO-PCR and MTPA-PCR methods for genotyping of rs267729 (C>G) polymorphism was found. MTPA-PCR could be a reliable, accurate and simple technique for genotyping SNP and different mutations. This work describes a rapid, relatively cheap, high throughput detection of C>G polymorphism in ADIPOQ that can be used in large scale clinical studies. So far, no study was done on optimization methods for genotyping rs267729 (C>G) polymorphism by MTPA-PCR. Here, we successfully adjusted and enhanced this method for recognizing rs267729 (C>G) polymorphism in the promoter of ADIPOQ gene in Argentinian population.

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