Designing a Rapid Screening Method for Detection of Four ‎Common Beta Globin Gene Mutations in Iranian Population by ‎High-Resolution Melting Analysis

Aims: Beta-thalassemia is a blood and genetic disorder. Prevalence of beta-thalassemia trait is high in Iranian population. Therefore, for detection of carriers’ status in premarital screening we need to establish reliable and rapid scanning method. The aim of this study is development a rapid method to detect four common beta globin gene mutations in Iranian population by HRM (High-Resolution Melting Analysis.
In this experimental study, eight samples of beta-thalassemic carriers and illness with IVSI-110 (G>A), IVSII-I (G>A), IVSI-5 (G>C) and CD36/37 (-T) genotypes in Homozygote and heterozygote conditions and one control sample (healthy person) were selected. After blood collection, DNA extraction was performed using salting out method. To ensure, the samples were run binary, and two control samples without DNA were used. Primers were designed with Gene Runner 6.0.28, Primer Express v 3.0.1 3/1) and Primer3 v. 0.4.0 and HRM method was used to determine the above mutations.
Findings: The best concentration of DNA was obtained at 5ng/μl. In the difference plot step, the differences between the curves were clearly observed in heterozygous, normal and homozygous conditions. Unexpectedly, in the mutation IVSII-IG>A in the melting curve was seen two peaks in the samples; it was due to the presence of a polymorphism near the desired mutation.
The HRM method is capable of detecting the four common beta globin gene mutations, and, it can differentiate between heterozygote and homozygous in a single mutation. This method is high throughput, which can analyze 96 samples of DNA in 2hours.