An Optimized Electrophoresis System for Tandem SSCP and Heteroduplex Analysis of p53 Gene Exons 5-9 on Glass Microfluidic Chips
Christa N. Hestekin, J P. Jakupciak, Thomas N. Chiesl, C D. O'Connell, Annelise E. Barron, C W. Kan
With the sequencing of the human genome, there is a growing need for rapid and sensitive genotyping methods that can be incorporated into the clinical setting. DNA-based methods, such as single strand conformational polymorphism (SSCP) and heteroduplex analysis (HA), are commonly used in a research setting to examine mutations in cancer-related genes, but need optimization to achieve the high sensitivity needed for a clinical setting. Here we investigate the importance of several parameters such as polymer matrix, wall coating, and electric field strength on the mutation detection and sensitivity of microchip electrophoresis-SSCP/HA for exons 5-9 of the p53 gene. By looking at the effect of concentration and molecular weight of the polymer matrix, linear polyacrylamide, we determined that 8% 600 kDa was the optimum polymer for providing high resolution of the mutation detection. In addition, we found that including a small amount of the polymer wall coating, poly-N-hydroxyethylacrylamide, improved resolution, decreased loading times, and extended coating lifetime.
, Jakupciak, J.
, Chiesl, T.
, O'Connell, C.
, Barron, A.
and Kan, C.
An Optimized Electrophoresis System for Tandem SSCP and Heteroduplex Analysis of p53 Gene Exons 5-9 on Glass Microfluidic Chips, Electrophoresis
(Accessed September 29, 2023)