Detection of α-thalassemias by multiplex polymerase chain reaction

Abstract

Although α-thalassemia is the most common genetic abnormality in the world, there is currently no routine laboratory method to definitively identify individuals who are affected. We describe a rapid and simple method that utilizes deletion-sensitive primers to amplify normal DNA sequences. Deletions involving the regions responsible for most of the α-thalassemia cases in the US prevent amplification with these primers. In tests with DNA isolated from small amounts (10 μL) of whole blood, the deletion-sensitive primers gave rise to the expected 248- and 375-bp (base pair) amplification products in normal individuals. These primers, along with primers designed to bind to a nonaffected control sequence from the hemoglobin β chain, could be amplified simultaneously (multiplex polymerase chain reaction). This made it possible to detect heterozygotes for α-thalassemia-2 (one α locus deleted) by determining the ratios of the 248- and 375-bp amplification products to the product of the control sequence (268 bp). The method is rapid and simple and can be performed in a routine clinical laboratory.