Multiplex minisequencing screen for common Southeast Asian and Indian beta-thalassemia mutations.
BACKGROUND
Beta-thalassemia is endemic to many regions in Southeast Asia and India, and <20 beta-globin gene mutations account for > or =90% of beta-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 beta-globin gene from genomic DNA and to detect the Delta619bp deletion mutation. Multiplex minisequencing was then performed on the amplified beta-globin fragment to detect an additional 15 common Southeast Asian and Indian beta-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 beta-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 beta-thalassemia mutations, is amenable to high-throughput scale up, and may bring population-based screening of beta-thalassemia in endemic regions a step closer to implementation.
Wang
Wen
W
Department of Pediatrics, National University of Singapore, Singapore 119074, Singapore.
Kham
Shirley K Y
SK
Yeo
Gare-Hoon
GH
Quah
Thuan-Chong
TC
Chong
Samuel S
SS
D001210:Asia, Southeastern; D001322:Autoanalysis; D019075:Electrophoresis, Capillary; D005838:Genotype; D005914:Globins / Q000235:genetics; D006801:Humans; D007194:India; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D017086:beta-Thalassemia / Q000235:genetics*
D005914:Globins