Method Development of Nested Allele-Specific Multiplex Polymerase Chain Reaction (NASM-PCR) for the Irritable Bowel Syndrome (IBS)-related Gene Polymorphisms

Authors

  • Rasmaizatul Akma Rosdi School of Health Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia https://orcid.org/0000-0002-0630-9303
  • Nurfadhlina Musa Human Genome Center, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia https://orcid.org/0000-0002-7533-8474
  • Zalina Zahari Faculty of Pharmacy, Universiti Sultan Zainal Abidin, Besut Campus, 22200 Besut, Terengganu, Malaysia https://orcid.org/0000-0003-1459-8958
  • Mohd Khairi Zahri Faculty of Health Sciences, Universiti Sultan Zainal Abidin, Gong Badak Campus, 21300 Kuala Nerus, Terengganu, Malaysia https://orcid.org/0000-0002-6582-6001
  • Mulham Alfatama Faculty of Pharmacy, Universiti Sultan Zainal Abidin, Besut Campus, 22200 Besut, Terengganu, Malaysia https://orcid.org/0000-0003-4592-7064
  • Boon Yin Khoo Institute for Research in Molecular Medicine, Universiti Sains Malaysia, 11800 Gelugor, Penang, Malaysia https://orcid.org/0000-0003-1915-6606

DOI:

https://doi.org/10.24996/ijs.2023.64.3.9

Keywords:

Irritable bowel syndrome (IBS), gastrointestinal (GI) tract, nested allele-specific multiplex PCR, genotyping method, single nucleotide polymorphism (SNP)

Abstract

Polymorphisms in the genes of G-protein subunit beta 3 (GNB3); rs5443, tryptophan hydroxylase 1 (TPH1); rs211105 and rs4537731, tryptophan hydroxylase 2 (TPH2); rs4570625 and sodium voltage-gated channel alpha subunit 5 (SCN5A); rs1805124, have known to cause the abnormalities in the gastrointestinal tract that are implicated to irritable bowel syndrome (IBS) predisposition. Upfront genetic polymorphism genotyping in IBS-related gene polymorphisms will help to intervene and guide the decision-making in the management of IBS patients. This study aimed to develop a genotyping method to detect the respective polymorphisms using nested allele-specific multiplex polymerase chain reaction (NASM-PCR). A combination of nested and allele-specific multiplex PCR method was developed to determine the five single nucleotide polymorphisms (SNPs) mentioned. Annealing temperature, annealing and extension times, and the concentrations of MgCl2, primers, and DNA samples were optimized in the PCR. Sanger sequencing was performed to validate the genotyping results. NASM-PCR for IBS-related gene polymorphisms were successfully developed. DNA bands which correspond to the genes and SNPs have shown 100% homologous with the gene database. The developed method of NASM-PCR was reproducible and specific to be used for determining the respective polymorphisms of IBS. Notably, the described method can easily be integrated into other laboratories for population study or clinical use.

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Published

2023-02-28

Issue

Section

Biotechnology

How to Cite

Method Development of Nested Allele-Specific Multiplex Polymerase Chain Reaction (NASM-PCR) for the Irritable Bowel Syndrome (IBS)-related Gene Polymorphisms. (2023). Iraqi Journal of Science, 64(3), 1129-1141. https://doi.org/10.24996/ijs.2023.64.3.9

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