Multi-allele DNA biosensor for the rapid genotyping of 'nondeletion' alpha thalassaemia mutations in HBA1 and HBA2 genes by means of multiplex primer extension reaction.

Background: Alpha-thalassaemia is an autosomal recessive disorder characterized by defective production of the alpha chain of haemoglobin. It is caused mainly by deletions of one or both of the duplicated alpha-globin genes on chromosome 16, and/or by nucleotide variations, known as "nondeletion" mutations. Definition of the alpha globin genotype in carriers supports genetic counselling, and in patients with Hb H disease is useful to predict prognosis and management options. Here, we report a method that facilitates direct detection by naked eye of the 13 most common "nondeletion" alpha-globin gene mutations in populations around the Mediterranean and Middle East.

Methods And Results: The method comprises (i) PCR amplification of a single 1087 bp fragment for each HBA1 and HBA2 gene (separately); (ii) multiplex primer extension reaction of just 10 cycles, using unpurified amplification product as a template, to incorporate biotin into those allele-specific primers that extend and, finally, (iii) visual detection of the reaction products within minutes by the dipstick biosensor. The method was evaluated by analysing 105 samples of known genotypes and the results were found fully concordant with those obtained by the reference methods.

Conclusions: The proposed assay is particularly suited for small molecular-diagnostic laboratories with a limited budget and a low-to-medium sample volume. In addition this platform represents a very simple and useful genotyping tool to support gene scanning methods whenever nucleotide variations have to be specified.