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Development and application of low-cost T-ARMS-PCR assay for AGT and CYP11B1 gene polymorphisms

  • Misbah Hussain
  • Haq Nawaz Khan
  • Fazli Rabbi AwanEmail author
Original Article
  • 27 Downloads

Abstract

Angiotensin II (Ang II: a truncated octapeptide of angiotensinogen, AGT) and 11-β-hydroxylase influence regulation of blood pressure. Dysregulation of Ang II and 11-β-hydroxylase can lead to hypertension and elevate aldosterone levels. Polymorphisms in AGT (encodes AGT) and CYP11B1 (encodes 11-β-hydroxylase) shift the paradigm from physiological to pathological. Currently, various high-throughput techniques are used to genotype these polymorphisms. These techniques require expensive infrastructure and reagents. However, in developing countries, where cost is the main limiting factor, it is not feasible to use expensive techniques. So, the aim of current study was to develop efficient low-cost method for genotyping of cardiovascular disease and hypertension associated polymorphisms of AGT (rs4762, rs5051) and CYP11B1 (rs6410). For this, tetra amplification-refractory mutation system-polymerase chain reaction (T-ARMS-PCR) method was developed and optimized for aforementioned AGT and CYP11B1 gene polymorphisms. Efficiency of T-ARMS-PCR was tested by genotyping 776 human samples. These T-ARMS-PCR assays were also validated by Sanger DNA sequencing, where 100% concordance was found, allowing the efficient use of these T-ARMS-PCR assays for polymorphism genotyping in AGT and CYP11B1 in resource limited settings. T-ARMS-PCR is low-cost, efficient and reliable assay for genotyping of AGT and CYP11B1 gene polymorphisms.

Keywords

PCR assay T-ARMS-PCR Genotyping RAAS AGT CYP11B1 

Notes

Funding

This study was supported by the Higher Education Commission (HEC), Pakistan.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This study was approved by the institutional (National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan) ethics review committee.

Human and animal rights

All procedures were in accordance with the guidelines of Declaration of Helsinki for working with human subjects.

Informed consent

Informed consent was obtained from all individual participants included in this study.

Supplementary material

11033_2018_4493_MOESM1_ESM.docx (20 kb)
Supplementary material 1 (DOCX 19 KB)

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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Diabetes and Cardio-Metabolic Disorders Laboratory, Health Biotechnology DivisionNational Institute for Biotechnology and Genetic Engineering (NIBGE)FaisalabadPakistan
  2. 2.Pakistan Institute of Engineering and Applied Sciences (PIEAS)NilorePakistan

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