Quantitation of mitochondrial DNA A1555G mutation by real time amplification refractory mutation system quantitative PCR

Abstract

Objective To develop a real time amplification refractory system(RT-ARMS-qPCR) quantitative PCR method with SYBR Green I to assess the mtDNA A1555G mutation. Methods A specific fragment flanking mtDNA 1555 site was amplified with PCR and ligated into a pGEM Easy T vector. Serial dilutions of the plasmid DNA were quantified the actual copy numbers were assessed using RF-ARMS-qPCR with SYBR Green I. RF-ARMS-qPCR was established with mismatched base pairs at 3' in the primer todetect the copy number of mtDNA containing wild or mutant mtDNA. The specificity of amplified products was checked by melting curve analysis. Results The intra- and interassay variation was 1.34% and 1.96%, respectively when the assay was used to detect 1 copy/ul recombinant template of plasmid. Thequantitative standard curve showed that the assay had good linear correlation from 102 copies/ul to108 copies/ul. This assay could be served for the quantification of other samples. There was significantcorrelation between frequency of mutant mtDNA and phenotype (r=0.771, P = 0.003) in hearing lossgroup. Conclusions The established assay can be used to detect quantitatively mtDNA A1555G mutation byRF-ARMS-qPCR. This assay is specific, stable and accurate. There is significant correlation betweenquantification of mtDNA AI555G and the severity of hearing loss. Key words: Hearing impaired persons; DNA, mitochondrial; Point mutation