Detection of Mutations in the Dystrophin Gene via Automated DHPLC Screening and Direct Sequencing

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Detection of Mutations in the Dystrophin Gene via Automated DHPLC Screening and Direct Sequencing

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Title: Detection of Mutations in the Dystrophin Gene via Automated DHPLC Screening and Direct Sequencing
Author: Bennett, Richard R; den Dunnen, Johan; O'Brien, Kristine F; Darras, Basil T.; Kunkel, Louis Martens

Note: Order does not necessarily reflect citation order of authors.

Citation: Bennett, Richard R, Johan den Dunnen, Kristine F O'Brien, Basil T Darras, and Louis M Kunkel. 2001. Detection of mutations in the dystrophin gene via automated DHPLC screening and direct sequencing. BMC Genetics 2: 17.
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Abstract: Background: Currently molecular diagnostic laboratories focus only on the identification of large deletion and duplication mutations (spanning one exon or more) for Duchenne Muscular Dystrophy (DMD) yielding 65% of causative mutations. These mutations are detected by an existing set of multiplexed polymerase chain reaction (PCR) primer pairs. Due to the large size of the dystrophin gene (79 exons), finding point mutations (substitutions, deletions or insertions of one or several nucleotides) has been prohibitively expensive and laborious. The aim of this project was to develop an effective and convenient method of finding all, or most, mutations in the dystrophin gene with only a moderate increase in cost. Results: Using denaturing high performance liquid chromatography (DHPLC) screening and direct sequencing, 86 PCR amplicons of genomic DNA from the dystrophin gene were screened for mutations in eight patients diagnosed with DMD who had tested negative for large DNA rearragements. Mutations likely to be disease-causative were found in six of the eight patients. All 86 amplicons from the two patients in whom no likely disease-causative mutations were found were completely sequenced and only polymorphisms were found. Conclusions: We have shown that it is now feasible for clinical laboratories to begin testing for both point mutations and large deletions/duplications in the dystrophin gene. The detection rate will rise from 65% to greater than 92% with only a moderate increase in cost.
Published Version: doi://10.1186/1471-2156-2-17
Other Sources: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC59832/pdf/
Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA
Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:4873531

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