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Departments of
1
Pediatrics and
2
Obstetrics & Gynecology, National University of Singapore, Singapore 119074, Singapore.
3 The Children’s Medical Institute and
4
Molecular Diagnosis Center, Department of Laboratory Medicine, National University Hospital, Singapore 119074, Singapore.
5 Departments of Pediatrics and of Gynecology &Obstetrics, The Johns Hopkins University School of Medicine, Baltimore, MD 21287.
aAddress correspondence to this author at: Department of Pediatrics, National University of Singapore, Level 4, National University Hospital, 5 Lower Kent Ridge Rd., Singapore 119074, Singapore. Fax 65-6779-7486; e-mail paecs{at}nus.edu.sg.
Background: ß-Thalassemia is endemic to many regions in Southeast Asia and India, and <20 ß-globin gene mutations account for 
90% of ß-thalassemia alleles in these places. We describe a multiplex minisequencing assay to detect these common mutations.
Methods: Gap-PCR was used to simultaneously amplify the ß-globin gene from genomic DNA and to detect the 
619bp deletion mutation. Multiplex minisequencing was then performed on the amplified ß-globin fragment to detect an additional 15 common Southeast Asian and Indian ß-thalassemia mutations. Site-specific primers of different lengths were subjected to multiple rounds of annealing and single-nucleotide extension in the presence of thermostable DNA polymerase and the four dideoxynucleotides, each labeled with a different fluorophore. Minisequencing products were separated and detected by capillary electrophoresis, followed by automated genotyping. The optimized assay was subjected to a double-blind validation analysis of 89 ß-thalassemia and wild-type DNA samples of known genotype.
Results: Homozygous wild-type or mutant DNA samples produced electropherograms containing only a single colored peak for each mutation site, whereas samples heterozygous for a specific mutation displayed two different-colored peaks for that mutation site. Samples were automatically genotyped based on color and position of primer peaks in the electropherogram. In the double-blind validation analysis, all 89 DNA samples were genotyped correctly (100% assay specificity).
Conclusions: The described semiautomated multiplex minisequencing assay can detect the most common Southeast Asian and Indian ß-thalassemia mutations, is amenable to high-throughput scale up, and may bring population-based screening of ß-thalassemia in endemic regions a step closer to implementation.
The following articles in journals at HighWire Press have cited this article:
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  K. Glynou, P. Kastanis, S. Boukouvala, V. Tsaoussis, P. C. Ioannou, T. K. Christopoulos, J. Traeger-Synodinos, and E. Kanavakis High-Throughput Microtiter Well-Based Chemiluminometric Genotyping of 15 HBB Gene Mutations in a Dry-Reagent Format Clin. Chem., March 1, 2007; 53(3): 384 - 391. [Abstract] [Full Text] [PDF]
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 E. P. Vichinsky, E. A. MacKlin, J. S. Waye, F. Lorey, and N. F. Olivieri Changes in the Epidemiology of Thalassemia in North America: A New Minority Disease Pediatrics, December 1, 2005; 116(6): e818 - e825. [Abstract] [Full Text] [PDF]
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S. P. Yip, S. F. Pun, K. H. Leung, and S. Y. Lee Rapid, Simultaneous Genotyping of Five Common Southeast Asian {beta}-Thalassemia Mutations by Multiplex Minisequencing and Denaturing HPLC Clin. Chem., October 1, 2003; 49(10): 1656 - 1659. [Full Text] [PDF]
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W. Wang, E. S.K. Ma, A. Y.Y. Chan, D. H.K. Chui, and S. S. Chong Multiple Minisequencing Screen for Seven Southeast Asian Nondeletional {alpha}-Thalassemia Mutations Clin. Chem., May 1, 2003; 49(5): 800 - 803. [Full Text] [PDF]
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