title: Design and validation of a conformation-sensitive capillary electrophoresis system for mutation identification of the COL7A1 gene with automated peak comparison.
abstract: Dystrophic epidermolysis bullosa is a heritable skin disease in which blisters occur because of a defect in type VII collagen resulting from mutations in the COL7A1 gene that is composed of 118 exons. Although a few mutations are specific to certain populations owing to founder effects, and although a few mutational hotspots exist, most mutations are unique to families and can be found scattered throughout the entire COL7A1 gene. This emphasizes the need for a sensitive, reliable, and efficient mutation scanning technique. Therefore, we developed a conformation-sensitive capillary electrophoresis (CSCE) system for COL7A1 mutation scanning. Here we report on the design and validation of this system. The CSCE technique is based on the principle of heteroduplex formation when polymerase chain reaction-amplified DNA fragments containing heterozygous sequence changes are slowly reannealed. These fluorescently labeled fragments have different migration characteristics and can be detected on a multi-capillary automated sequencer. Validation was performed by analysis of 29 known COL7A1 sequence changes, covering 33% of amplicons. After optimization of the conditions, all 29 sequence changes were detected by the CSCE system, irrespective of length or CG-content of amplicons and position of sequence changes, reflecting an analytical sensitivity of 90.2-100% (95% confidence interval). We conclude that this CSCE system is a rapid, reliable, cost-effective, and highly sensitive way of mutation scanning for COL7A1 in a molecular genetics service laboratory.
authors.affiliation: Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. p.c.van.den.akker@medgen.umcg.nl
authors.forename: Peter C
authors.initials: PC
authors.lastname: van den Akker
authors.forename: Wendy
authors.initials: W
authors.lastname: Hettema
authors.forename: Rowdy
authors.initials: R
authors.lastname: Meijer
authors.forename: Marcel F
authors.initials: MF
authors.lastname: Jonkman
authors.forename: Robert M W
authors.initials: RM
authors.lastname: Hofstra
authors.forename: Hans
authors.initials: H
authors.lastname: Scheffer
mesh_terms: D001483:Base Sequence; D003094:Collagen / Q000235:genetics*; D017931:DNA Primers; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction
chemical_list: D017931:DNA Primers; D005456:Fluorescent Dyes; D003094:Collagen