A Rapid Multiplex ARMS-PCR Method for the Detection of Four Single Nucleotide Polymorphisms of the Vitamin D Receptor (VDR) Gene

The most studied single nucleotide polymorphisms of the VDR gene are Bsm1, Apa1, Taq1 and Fok1. Previously, many approaches have been used to study SNPs in VDR gene including restriction fragment length polymorphism (RFLP), Single Amplification refractory mutation system PCR (Single-ARMS-PCR), and sequencing of the VDR gene. The objective of the study was to develop a multiplex ARMS-PCR system for genotyping the four SNPs of the Vitamin D Receptor gene in just two simultaneous reactions. DNA samples of 353 individuals were genotyped for Bsm1, Apa1, Taq1 and Fok1 of the VDR gene SNPs using the MultiplexARMS-PCR system. Thirty two DNA samples were randomly selected and genotyped for the same polymorphisms with the Single-ARMS-PCR method, while genotyping with the Multiplex-ARMS-PCR was repeated for 37 DNA samples to verify for reproducibility. The Multiplex-ARMS-PCR system gave consistent results with the Single-ARMS-PCR system. The genotype frequencies of the Fok1 ploymorphism were FF (67.1%), Ff (28.9%) and ff (4.0%); for Bsm1 were BB (9.4%), Bb (38.8%) and bb (51.8%); Taq1 were TT (46.7%), Tt (47.3%) and tt (6.0%) and for Apa1 were AA (42.8%), Aa (40.5%) and aa (16.7%). The allelic frequency of ‘F’ versus ‘f’ was 82% versus 18%, ‘B’ versus ‘b’ was 29% versus 71%, ‘T’ versus ‘t’ was 70% versus 30% and ‘A’ versus ‘a’ was 63% versus 37%. The MultiplexARMS-PCR technique gave consistent results throughout the study and was comparable when repeated separately using Single-ARMS-PCR method. This technique was found to be simple, reliable and less time consuming, thus could be a useful alternative to study VDR gene polymorphisms in routine clinical and resource-limited settings.