Rapid and Simultaneous Detection of Vitamin D Receptor Gene Polymorphisms by a Single ARMS-PCR Assay
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Rapid and Simultaneous Detection of Vitamin D Receptor Gene Polymorphisms by a Single ARMS-PCR Assay Mohammad Jafari • Aliyar Pirouzi • Saber Anoosheh • Parisa Farnia • Nader Tajik
Ó Springer International Publishing Switzerland 2013
Abstract Background Vitamin D has various roles in many biological actions such as calcium homeostasis, cell proliferation, and cell differentiation to many target tissues. These effects are mediated by the active form of vitamin D, 1,25(OH)2D3, which binds to a cytoplasmic protein called vitamin D receptor (VDR). VDR gene has four common single nucleotide polymorphisms (SNPs) that are defined by the presence of restriction sites for FokI (F/f), TaqI (T/ t), BsmI (B/b), and ApaI (A/a). The association of VDR gene polymorphisms with several diseases has been investigated. In most studies, VDR genotyping was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assays, which are cumbersome and time consuming, and their results are sometimes difficult to interpret. Objective We modified previously reported primers for VDR genotyping and set up a single amplification-
refractory mutation system (ARMS)-PCR method for simultaneous genotyping of four common VDR polymorphisms. Methods In this study, 218 DNA samples were analyzed for VDR genetic variants by this ARMS-PCR technique; 136 of them were re-genotyped by PCR-RFLP assays to compare genotyping results. Result We obtained allelic frequencies of 69 vs. 31 % for F/f, 34 vs. 66 % for B/b, 70 vs. 30 % for T/t, and 52 vs. 48 % for A/a in this sample of the Iranian population. In addition, comparisons of the results of these two methods showed good uniformity in VDR genotypes; although, in some samples, ambiguity in restriction patterns was present. Conclusion As ARMS-PCR is more rapid, economic, and user friendly than PCR-RFLP, its substitution would be welcomed in disease association and pharmacogenetic studies of VDR variants.
M. Jafari A. Pirouzi Gerash Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
1 Introduction
M. Jafari A. Pirouzi N. Tajik (&) Division of Transplant Immunology and Immunogenetics, Department of Immunology, School of Medicine, Iran University of Medical Sciences, Shahid Hemmat Highway, 14496 Tehran, Iran e-mail: [email protected] S. Anoosheh Department of Molecular Medicine, University of Padova, Padua, Italy S. Anoosheh P. Farnia Mycobacteriology Research Centre, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran
Vitamin D has roles in a variety of biological actions, such as calcium homeostasis, and many non-classical actions. The non-classical actions of vitamin D can be categorized into three general effects: regulation of hormone secretion, regulation of immune function, and regulation of cellular proliferation and differentiation [1]. The ability of 1,25(OH)2D3, the active metabolite of vitamin D, to regulate hormone secretion plays an
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