Rapid and sensitive p53 mutation detection by microchip electrophoresis-based tandem single strand conformation polymorphism/heteroduplex analysis

Genetic mutation detection technologies promise to revolutionize the diagnosis and treatment of cancer and other genetic diseases by enabling the correlation of prognosis with specific sequence alterations in tumor DNA. Mutations in the p53 gene, in particular, are important in the pathogenesis of human cancers and are challenging to detect.; Single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are two excellent, complementary electrophoretic methods for genetic mutation detection, which offer simplicity, broad applicability, and low cost. In SSCP, mutant DNA is mixed with wild-type DNA, denatured, and snap-cooled so that in dilute solutions, single-stranded DNA conformers form. Changes in DNA sequence lead to different conformations, which can be separated by electrophoresis in gels or entangled polymer media. In HA, mutant DNA is mixed with wild-type DNA, denatured, and cooled slowly, allowing some mutant and wild-type single strands of DNA to anneal. Sequence non-complementarities in "heteroduplexes" between the hybridized mutant and wild-type DNA may lead to conformational differences relative to wild-type and homoduplexes, which in some cases are separable by gel electrophoresis.; In order to create a low-cost, clinically feasible mutation detection system, we have implemented a novel, tandem-SSCP/HA method on microfluidic electrophoresis chips. We have investigated variables such as the polymer separation matrix properties, polymer wall coating, electric field strength, DNA sample stability and purification, temperature of analysis and the percentage of mutant DNA. This project involved significant work in the areas of polymer synthesis and characterization, molecular biology, and bioanalytical technology development. We demonstrate the ability of our new system to detect single-base mutations in the p53 gene exons 5-9 by microchip electrophoresis-SSCP/HA with separation times less than 10 minutes. The most important parameters for optimization were the concentration and molecular weight of the polymer matrix, the hydrophilicity of the polymer wall coating, and analysis temperature. Microchip electrophoresis-SSCP/HA is a highly robust method for genetic screening. A blinded study of 106 p53 exon 5-9 samples further demonstrated that microchip electrophoresis-SSCP/HA can provide 98% sensitivity and specificity of genetic mutation detection. The method was piloted on patient tumor samples and shows promise as a clinically applicable system for mutation detection.