title: Toward a fluorescent single-strand conformation polymorphism technique that detects all mutations: F-DOVAM-S.
abstract: Although DOVAM-S (detection of virtually all mutations-SSCP) in effect detects all mutations and is less costly than direct sequencing, the technique currently requires the use of radioactivity. F-DOVAM-S (fluorescent DOVAM-S) was developed to replace the isotopic label with fluorescence and to increase throughput via dye color multiplexing. As proof of principle, two multitemperature slab gel electrophoresis conditions were evaluated through the blinded analysis of mutations in the factor IX (FIX) genes of 88 hemophilia B (HB) patients and 7 wild-type controls. Using only two conditions, it was determined that F-DOVAM-S had a detection sensitivity of 97%. It is anticipated that when three or four optimized conditions are employed, F-DOVAM-S will detect all mutations. Three patient samples were multiplexed per well using three different fluorescent dyes (6FAM, VIC, and NED), demonstrating that it is possible to analyze up to 44 kb of diploid, color-coded amplification product per gel lane. This value corresponds to a throughput of approximately 4 Mb of DNA analyzed per 96-well gel, which is approximately triple that of conventional radiolabeled DOVAM-S. Throughput is further enhanced by the rapidity at which the fluorescent signal can be captured and the resultant multicolor chromatograms analyzed. Given these data, F-DOVAM-S has the potential to be a particularly powerful technology for clinical diagnosis because it allows the mutation analysis of multiple patients to be performed within 24h.
authors.affiliation: Department of Molecular Genetics and Department of Molecular Diagnosis, City of Hope National Medical Center, Duarte, CA 91010, USA.
authors.forename: Cameron
authors.initials: C
authors.lastname: Mroske
authors.forename: John
authors.initials: J
authors.lastname: Muci
authors.forename: Jicheng
authors.initials: J
authors.lastname: Wang
authors.forename: Kai
authors.initials: K
authors.lastname: Li
authors.forename: Wenjia
authors.initials: W
authors.lastname: Song
authors.forename: Jin
authors.initials: J
authors.lastname: Yan
authors.forename: Jinong
authors.initials: J
authors.lastname: Feng
authors.forename: Qiang
authors.initials: Q
authors.lastname: Liu
authors.forename: Steve S
authors.initials: SS
authors.lastname: Sommer
mesh_terms: D001483:Base Sequence; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D005164:Factor IX / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D018807:Polymorphism, Single-Stranded Conformational; D012680:Sensitivity and Specificity
chemical_list: D005456:Fluorescent Dyes; D005164:Factor IX