title: High-throughput microtiter well-based chemiluminometric genotyping of 15 HBB gene mutations in a dry-reagent format.
abstract: BACKGROUND
  Hemoglobinopathies are the most common inherited diseases worldwide. Various methods for genotyping of hemoglobin, beta (HBB) gene mutations have been reported, but there is need for a high sample-throughput, cost-effective method for simultaneous screening of several mutations. We report a method that combines the high detectability and dynamic range of chemiluminescence with the high allele-discrimination ability of probe extension reactions for simultaneous genotyping of 15 HBB mutations in a high sample-throughput, dry-reagent format.
  
  
  METHODS
  We genotyped the HBB mutations IVSI-110G>A, CD39C>T, IVSI-1G>A, IVSI-6T>C, IVSII-745C>G, IVSII-1G>A, FSC6GAG>G-G, -101C>T, FSC5CCT>C-, IVSI-5G>A, FSC8AAG>-G, -87C>G, IVSII-848C>A, term+6C>G, and HbS (cd6GAG>GTG). The method used comprises the following: (a) duplex PCR that produces fragments encompassing all 15 mutations, (b) probe extension reactions in the presence of fluorescein-modified dCTP, using unpurified amplicons, and (c) microtiter well-based assay of extension products with a peroxidase-antifluorescein conjugate and a chemiluminogenic substrate. We used lyophilized dry reagents to simplify the procedure and assigned the genotype by the signal ratio of the normal-to-mutant-specific probe.
  
  
  RESULTS
  We standardized the method by analyzing 60 samples with known genotypes and then validated by blindly genotyping 115 samples with 45 genotypes. The results were fully concordant with sequencing. The reproducibility (including PCR, probe extension reaction, and chemiluminometric assay) was studied for 20 days, and the CVs were 11%-19%.
  
  
  CONCLUSIONS
  This method is accurate, reproducible, and cost-effective in terms of equipment and reagents. The application of the method is simple, rapid, and robust. The microtiter well format allows genotyping of a large number of samples in parallel for several mutations.
authors.affiliation: Medicon Hellas S.A., Gerakas, Greece. glynou@mediconsa.com
authors.forename: Kyriaki
authors.initials: K
authors.lastname: Glynou
authors.forename: Petros
authors.initials: P
authors.lastname: Kastanis
authors.forename: Sotiria
authors.initials: S
authors.lastname: Boukouvala
authors.forename: Vassilis
authors.initials: V
authors.lastname: Tsaoussis
authors.forename: Penelope C
authors.initials: PC
authors.lastname: Ioannou
authors.forename: Theodore K
authors.initials: TK
authors.lastname: Christopoulos
authors.forename: Joanne
authors.initials: J
authors.lastname: Traeger-Synodinos
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Kanavakis
mesh_terms: D005828:Genetics, Population; D005838:Genotype; D005914:Globins / Q000235:genetics*; D006801:Humans; D008163:Luminescent Measurements; D009154:Mutation; D016133:Polymerase Chain Reaction; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D017086:beta-Thalassemia / Q000235:genetics*
chemical_list: D011933:Reagent Kits, Diagnostic; D005914:Globins