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This Article Full Text Full Text (PDF) 077776.Supplemental Data All Versions of this Article: clinchem.2006.077776v1 53/3/384    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Web of Science (3) Citing Articles via Google Scholar Google Scholar Articles by Glynou, K. Articles by Kanavakis, E. Search for Related Content PubMed PubMed Citation Articles by Glynou, K. Articles by Kanavakis, E. Related Collections Molecular Diagnostics and Genetics

High-Throughput Microtiter Well-Based Chemiluminometric Genotyping of 15 HBB Gene Mutations in a Dry-Reagent Format

¹ Medicon Hellas S.A., Gerakas, Greece.
² Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Athens, Greece.
³ Department of Chemistry, University of Patras, Patras, Greece.
⁴ Foundation for Research and Technology Hellas, Institute of Chemical Engineering and High Temperature Chemical Processes, Patras, Greece.
⁵ Department of Medical Genetics, University of Athens, St. Sophia’s Children’s Hospital, Athens, Greece.

^aAddress correspondence to this author at: Medicon Hellas S.A., R&D Department, 5-7 Melitona St., Gerakas 15344, Greece. Fax 30-2106-612-666; e-mail glynou{at}mediconsa.com.

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.