An optimized microchip electrophoresis system for mutation detection by tandem SSCP and heteroduplex analysis for p53 gene exons 5-9.
With the complete sequencing of the human genome, there is a growing need for rapid, highly sensitive genetic mutation detection methods suitable for clinical implementation. DNA-based diagnostics such as single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are commonly used in research laboratories to screen for mutations, but the slab gel electrophoresis (SGE) format is ill-suited for routine clinical use. The translation of these assays from SGE to microfluidic chips offers significant speed, cost, and sensitivity advantages; however, numerous parameters must be optimized to provide highly sensitive mutation detection. Here we present a methodical study of system parameters including polymer matrix, wall coating, analysis temperature, and electric field strengths on the effectiveness of mutation detection by tandem SSCP/HA for DNA samples from exons 5-9 of the p53 gene. The effects of polymer matrix concentration and average molar mass were studied for linear polyacrylamide (LPA) solutions. We determined that a matrix of 8% w/v 600 kDa LPA provides the most reliable SSCP/HA mutation detection on chips. The inclusion of a small amount of the dynamic wall-coating polymer poly-N-hydroxyethylacrylamide in the matrix substantially improves the resolution of SSCP conformers and extends the coating lifetime. We investigated electrophoresis temperatures between 17 and 35 degrees C and found that the lowest temperature accessible on our chip electrophoresis system gives the best condition for high sensitivity of the tandem SSCP/HA method, especially for the SSCP conformers. Finally, the use of electrical fields between 350 and 450 V/cm provided rapid separations (<10 min) with well-resolved DNA peaks for both SSCP and HA.
Hestekin
Christa N
CN
Department of Chemical and Biological Engineering, Northwestern University, Evanston, IL, USA.
Jakupciak
John P
JP
Chiesl
Thomas N
TN
Kan
Cheuk Wai
CW
O'Connell
Catherine D
CD
Barron
Annelise E
AE
D000180:Acrylic Resins / Q000737:chemistry; D004252:DNA Mutational Analysis / Q000379:methods* / Q000592:standards; D046209:Electrophoresis, Microchip / Q000592:standards*; D005091:Exons / Q000235:genetics; D016158:Genes, p53 / Q000235:genetics*; D020180:Heteroduplex Analysis / Q000379:methods*; D006801:Humans; D018807:Polymorphism, Single-Stranded Conformational; D013696:Temperature; D000099166:Polyacrylamides
D000180:Acrylic Resins; D000099166:Polyacrylamides