MULTIPLEX PCR BASED SINGLE NUCLEOTIDE POLYMORPHISMS ANALYSIS OF HBS POINT MUTATION

Abstract

Introduction: Sickle hemoglobin (HbS), an autosomal recessive hemoglobinopathy cause of Sickle cell disease (SCD), is widely sprayed around the globe affecting millions of people . SCD results from single nucleotide polymorphism (SNP) or point mutation causing amino acid substitution from Glutamic acid to Valine leads to sickled shape red blood cells. SNPs can be well studied by using allele-specific amplification (ASA) technique. Aims & Objective: To develop a simple, rapid, easy and accurate genotyping method for SNP analysis of SCD. Materials and methods: By performing different tests, a well characterized sample panel of 150 different types of samples was prepared. From this sample panel DNA was extracted and used for SNP-genotyping of SCD. Specific primers were used for performing monoplex PCR amplification of wild type allele (HbAA) and mutant allele (HbSS) were performed individually. By using the same primers multiplex PCR assay was experimented. Results and conclusion: This is a simple and low cost molecular method for the detection of point mutation and useful tool for the diagnosis of SCD. The entire analysis can be performed in one reaction mixture, which results in higher speed, higher accuracy, and the need for smaller samples. This technique might be of great value for genotyping of homozygous sickle cell patients (SS) and heterozygous sickle cell trait (AS). But we found one discrepancy with double heterozygous (sickle β-thalassaemia) samples. We were not able to differentiate sickle cell carrier state (AS) from the double heterozygous like sickle β-thalassaemia state. So we conclude that for simultaneous detection of thalassaemia along with sickle cell requires addition of more primers specific for thalassaemia mutation. In addition to this when two bands, one for wild type allele and second for mutant allele appears, care must be taken to conclude whether the person is a sickle cell carrier (AS) or having double heterozygous (sickle β-thalassaemia) like condition.