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 wildtype, 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.
ASJC Scopus subject areas
- Analytical Chemistry