title: High-throughput, high-sensitivity genetic mutation detection by tandem single-strand conformation polymorphism/heteroduplex analysis capillary array electrophoresis.
abstract: We present the first optimization of linear polyacrylamide (LPA)-based DNA separation matrixes for an automated tandem microchannel single-strand conformation polymorphism (SSCP)/heteroduplex analysis (HA) method, implemented in capillary arrays dynamically coated with poly(N-hydroxyethylacrylamide) (polyDuramide). An optimized protocol for sample preparation allowed both SSCP and HA species to be produced in one step in a single tube and distinguished in a single electrophoretic analysis. A simple, two-color fluorescent sample labeling and detection strategy enabled unambiguous identification of all DNA species in the electropherogram, both single- and double-stranded. Using these protocols and a panel of 11 p53 mutant DNA samples in comparison with wild-type, we employed high-throughput capillary array electrophoresis (CAE) to carry out a systematic and simultaneous optimization of LPA weight-average molar mass (Mw) and concentration for SSCP/HA peak separation. The combination of the optimized LPA matrix (6% LPA, Mw 600 kDa) and a hydrophilic, adsorbed polyDuramide wall coating was found to be essential for resolution of CAE-SSCP/HA peaks and yielded sensitive mutation detection in all 11 p53 samples initially studied. A larger set of 32 mutant DNA specimens was then analyzed using these optimized tandem CAE-SSCP/HA protocols and materials and yielded 100% sensitivity of mutation detection, whereas each individual method yielded lower sensitivity on its own (93% for SSCP and 75% for HA). This simple, highly sensitive tandem SSCP/HA mutation detection method should be easily translatable to electrophoretic analyses on microfluidic devices, due to the ease of the capillary coating protocol and the low viscosity of the matrix.
authors.affiliation: Department of Chemical Engineering, Northwestern University, Evanston, Illinois 60208, USA.
authors.forename: Igor V
authors.initials: IV
authors.lastname: Kourkine
authors.forename: Christa N
authors.initials: CN
authors.lastname: Hestekin
authors.forename: Brett A
authors.initials: BA
authors.lastname: Buchholz
authors.forename: Annelise E
authors.initials: AE
authors.lastname: Barron
mesh_terms: D019075:Electrophoresis, Capillary; D016158:Genes, p53; D006801:Humans; D007202:Indicators and Reagents; D009154:Mutation / Q000235:genetics*; D018807:Polymorphism, Single-Stranded Conformational
chemical_list: D007202:Indicators and Reagents