High throughput DNA sequence variant detection by conformation sensitive capillary electrophoresis and automated peak comparison

• Published in: Genomics (San Diego, Calif.) Vol. 87; no. 3; pp. 427 - 432
• Main Authors: Davies, Helen, Dicks, Ed, Stephens, Philip, Cox, Charles, Teague, Jon, Greenman, Chris, Bignell, Graham, O'Meara, Sarah, Edkins, Sarah, Parker, Adrian, Stevens, Claire, Menzies, Andrew, Blow, Matt, Bottomley, Bill, Dronsfield, Mark, Futreal, P. Andrew, Stratton, Michael R., Wooster, Richard
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.03.2006; Elsevier
• Subjects: Algorithms; Biological and medical sciences; DNA - chemistry; DNA - genetics; DNA Mutational Analysis - methods; DNA sequencing; Electrophoresis, Capillary - methods; Fundamental and applied biological sciences. Psychology; Genes. Genome; Genetics of eukaryotes. Biological and molecular evolution; Genome, Human; Heteroduplex; Humans; Molecular and cellular biology; Molecular genetics; Mutation detection; Nucleic Acid Conformation; Point Mutation - genetics; Polymerase Chain Reaction; Polymorphism, Single Nucleotide - genetics; Reproducibility of Results; Single-stranded confirmation polymorphism (SSCP); Heteroduplex; Single-stranded confirmation polymorphism (SSCP); Mutation detection; DNA sequencing; Variant; Automation; Capillary electrophoresis; DNA; Genomics; Method; Detection; Conformation
• ISSN: 0888-7543; 1089-8646
• DOI: 10.1016/j.ygeno.2005.11.008

We report the development of a heteroduplex-based mutation detection method using multicapillary automated sequencers, known as conformation-sensitive capillary electrophoresis (CSCE). Our optimized CSCE protocol detected 93 of 95 known base substitution sequence variants. Since the optimization of the method, we have analyzed 215 Mb of DNA and identified 3397 unique variants. An analysis of this data set indicates that the sensitivity of CSCE is above 95% in the central 56% of the average PCR product. To fully exploit the mutation detection capacity of this method, we have developed software, canplot, which automatically compares normal and test results to prioritize samples that are most likely to contain variants. Using multiple fluorescent dyes, CSCE has the capacity to screen over 2.2 Mb on one ABI3730 each day. Therefore this technique is suitable for projects where a rapid and sensitive DNA mutation detection system is required.