title: 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.
abstract: 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.
  
  
  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.
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens 15771, Greece.
authors.forename: Margarita
authors.initials: M
authors.lastname: Petropoulou
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens 15771, Greece.
authors.forename: Amalia
authors.initials: A
authors.lastname: Poula
authors.affiliation: Laboratory of Medical Genetics, Athens University, Athens 11527, Greece.
authors.forename: Jan
authors.initials: J
authors.lastname: Traeger-Synodinos
authors.affiliation: Laboratory of Medical Genetics, Athens University, Athens 11527, Greece.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Kanavakis
authors.affiliation: Department of Chemistry, University of Patras, Patras 26500, Greece; Foundation for Research and Technology Hellas, Institute of Chemical Engineering and High Temperature Chemical Processes (FORTH/ICE-HT), Patras 26504, Greece.
authors.forename: Theodore K
authors.initials: TK
authors.lastname: Christopoulos
authors.affiliation: Laboratory of Analytical Chemistry, Department of Chemistry, Athens University, Athens 15771, Greece. Electronic address: ioannou@chem.uoa.gr.
authors.forename: Penelope C
authors.initials: PC
authors.lastname: Ioannou
mesh_terms: D015374:Biosensing Techniques / Q000191:economics / Q000379:methods*; D017931:DNA Primers / Q000138:chemical synthesis / Q000235:genetics; D015870:Gene Expression; D005820:Genetic Testing; D005838:Genotype; D006442:Glycated Hemoglobin / Q000235:genetics*; D006443:Hemoglobin A2 / Q000235:genetics*; D006801:Humans; D019083:Mediterranean Region; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D055542:alpha-Globins / Q000032:analysis / Q000235:genetics*; D017085:alpha-Thalassemia / Q000175:diagnosis* / Q000235:genetics
chemical_list: D017931:DNA Primers; D006442:Glycated Hemoglobin; D006443:Hemoglobin A2; D055542:alpha-Globins
keywords: Genotyping; HBA1 and HBA2 genes; Multi-allele DNA biosensor; Nondeletion mutations; Primer extension reaction