¹DNA Laboratory, Genetics Centre, Guy's & St Thomas' NHS Foundation Trust, London, UK

Correspondence: Dr EJ Ashton, DNA Laboratory, Genetics Centre, Guy's & St Thomas' NHS Foundation Trust, 5th Floor Guy's Tower, Guy's Hospital, London, SE1 9RT, UK. Tel: +44 020 7188 1714; Fax: +44 020 7188 7273; E-mail: emma.ashton@gstt.nhs.uk

Received 10 May 2007; Revised 13 July 2007; Accepted 31 July 2007; Published online 29 August 2007.

Abstract

We have developed a technique to screen for gross deletions/duplications and point mutations using one streamlined approach. Fluorescent multiplex quantitative PCR is used to determine the copy number of each exon, followed by conformation sensitive capillary electrophoresis (CSCE) of the same PCR products on a multi-capillary genetic analyser. We have developed this technique to screen all 79 exons of one of the largest human genes currently known (dystrophin) using 12 multiplex PCR assays. A blind trial of 50 male and 50 female samples, in which 84 mutations had previously been found and characterized by other techniques, showed 100% sensitivity and specificity. We then applied this method to screen over 100 patient samples previously screened for deletions and duplications of 28 exons from the two hotspot regions. Our data show that combining a full deletion/duplication screen with CSCE will detect a mutation in 98% of Duchenne muscular dystrophy patients and 93% of Becker muscular dystrophy patients where the clinical diagnosis is certain. This technique is applicable to any gene and is particularly suited to mutation screening of large genes, decreasing the time taken for a complete gene screen for nearly all mutation types.