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An optimized microchip electrophoresis system for mutation detection by tandem SSCP and heteroduplex analysis for p53 gene exons 5–9
Author(s) -
Hestekin Christa N.,
Jakupciak John P.,
Chiesl Thomas N.,
Kan Cheuk Wai,
O'Connell Catherine D.,
Barron Annelise E.
Publication year - 2006
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.200600358
Subject(s) - single strand conformation polymorphism , heteroduplex , microbiology and biotechnology , exon , dna , mutation , electrophoresis , materials science , gene , chemistry , chromatography , genetics , biology
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°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.