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
ABSTRACT
Because of the lack of an effective and economical control strategy against malaria [the most devastating infectious disease in developing countries] Transmission-Blocking Vaccines [TBVs] concept has been raised in recent years, promising a more efficient way to malaria control. TBVs aim at interfering and/or blocking pathogen development within the vector, halting transmission to non-infected vertebrate host. Aminopeptidase N [APN] is one of the most potent proteins in parasite development in Anopheles malaria vectors, which is strongly co-localized with human malaria parasites in the mosquito midgut epithelium. Therefore, Aminopeptidase N is one of the best choices for a new TBV. In this study for the first time we used 3'-RACE to amplify APN gene in Anopheles stephensi [An.stephensi], a major malaria vector in Iran, Indian subcontinent up to China by using different sets of primers including exon junction, conserved and specific region primers. Full length of APN was sequenced stepwise, which could be applied in designing a new regional TBV and act as an essential component of malaria elimination program in An. stephensi distribution areas. Primers design and method modification should be set up exactly in approach based amplifications. From results we came to this conclusion that that 3'-RACE could be applied to amplified key regions which are be-yond reach