Development of a high-resolution melting analysis method based on SYBR green-I for rs7216389 locus genotyping in asthmatic child patients

ABSTRACT

Asthma is caused by the combination of different factors. Current concepts of asthma pathogenesis emphasize on gene-environment interactions. Mega-genome scanning projects revealed that different Single Nucleotide Polymorphisms [SNPs] are related to asthma susceptibility. rs7216389-T is one of them that is related to childhood asthma and its effect on childhood asthma severity has been proved in different nations, however no study has been performed in Eastern Mediterranean and Middle East countries yet. To perform population genetic studies, a rapid and high-throughput screening method is necessary. High-resolution melting analysis is a rapid, powerful and accurate method, which is suitable for this type of studies. Therefore, it has been decided to develop a high-resolution melting method for rs7216389 locus genotyping in Iranian asthmatic children. In the current study, a high-resolution melting analysis method based on SYBR Green-I was developed to check the frequency of rs7216389-T mutation in Iranian asthmatic children for the first time. Second and third classes of intercalating dyes are commonly used for high-resolution melting method. However, in this study, SYBR Green-I was used for rs7216389 locus genotyping for the first time. Our results for 60 samples showed that SYBR Green-I has good efficacy for rs7216389 locus genotyping through high-resolution melting method in comparison with PCR-RFLP and sequencing. Comparison of our results based on HRM analysis with PCR-RFLP showed that our developed method is rapid, accurate, high-throughput and economic to study the rs7216389 locus in asthmatic children and it is applicable for other similar population genetic studies