title: Automated mutation screening using dideoxy fingerprinting and capillary array electrophoresis.
abstract: The rapid progress in the isolation of genes associated with human disease has resulted in an increasing demand for mutation screening methods. The molecular diagnosis of the long QT syndrome (LQTS), a cardiac disorder characterized by prolongation of the QT(c) interval in the ECG, syncopes, and sudden death, requires mutation screening of all exons in at least five genes, encoding cardiac Na(+) and K(+) channel subunits. A method for automated dideoxy fingerprinting (ddF) using capillary array electrophoresis (CAE) was developed and the efficiency of the method was tested by analyzing 24 DNA samples with mutations in one of the genes KCNQ1 and KCNH2, which are involved in 50% of LQTS cases. One of these mutations, 362insQK in KCNQ1, is novel. The sensitivity was 100% using a single electrophoresis temperature of 18 degrees C or 25 degrees C. However, analysis of the samples in both the sense and anti-sense direction were required for high sensitivity. Analysis in a single direction resulted in a decrease of the sensitivity to 74% and 70%, respectively. The throughput of the ddF method, if performed with a 16 capillary CAE instrument, is 288 samples per seven hr if each sample is analyzed on both strands.
authors.affiliation: Department of Clinical Biochemistry, Statens Serum Institut, Copenhagen, Denmark. psa@ssi.dk
authors.forename: L A
authors.initials: LA
authors.lastname: Larsen
authors.forename: M
authors.initials: M
authors.lastname: Johnson
authors.forename: C
authors.initials: C
authors.lastname: Brown
authors.forename: M
authors.initials: M
authors.lastname: Christiansen
authors.forename: R
authors.initials: R
authors.lastname: Frank-Hansen
authors.forename: J
authors.initials: J
authors.lastname: Vuust
authors.forename: P S
authors.initials: PS
authors.lastname: Andersen
mesh_terms: D001331:Automation / Q000379:methods; D027682:Cation Transport Proteins; D016172:DNA Fingerprinting / Q000379:methods*; D004252:DNA Mutational Analysis / Q000379:methods; D004268:DNA-Binding Proteins; D015224:Dideoxynucleosides / Q000378:metabolism*; D000072237:ERG1 Potassium Channel; D019075:Electrophoresis, Capillary / Q000379:methods*; D051638:Ether-A-Go-Go Potassium Channels; D005091:Exons / Q000235:genetics; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D007267:Injections; D007438:Introns / Q000235:genetics; D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; D008133:Long QT Syndrome / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D018807:Polymorphism, Single-Stranded Conformational; D015221:Potassium Channels / Q000235:genetics*; D024642:Potassium Channels, Voltage-Gated; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG
chemical_list: D027682:Cation Transport Proteins; D004268:DNA-Binding Proteins; D015224:Dideoxynucleosides; C072312:ERG protein, human; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; C092509:KCNH6 protein, human; D051656:KCNQ Potassium Channels; D051657:KCNQ1 Potassium Channel; C496909:KCNQ1 protein, human; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG