Detection of HB E by restriction site analysis of PCR-amplified DNA fragments

Abstract

There are many tests to detect haemoglobin E (Hb E) disease however interpretation may be difficult because Hb E co-migrates with Hb A2 in conventional electrophoresis or HPLC method. Here we report a simple and rapid technique for the identification of patients with Hb E disease by visual examination of restriction enzyme patterns of Mnl 1 digestion on a 0.63 kb DNA fragment. The method includes Polymerase Chain Reaction (PCR) by using two specific primers S1 and S3. In Hb E disease, Mnl 1 produces restriction fragment patterns that can distinguish these cases from normals. A 230bp DNA fragments were was found to be associated with the mutant locus, whereas 171 bp and 60 bp fragments were generated from the normal loci has improved discriminatory power in the genotype analysis of Hb E mutations. We studied 10 patients with Hb E disease from unrelated Malaysian patients. From our results Mnl 1 detected one patient being homozygous and 9 patients were heterozygous.