Multiplexed p53 Mutation Detection by Free-Solution Conjugate Microchannel Electrophoresis with Polyamide Drag-Tags Robert J. Meagher, †,§ Jennifer A. Coyne, † Christa N. Hestekin, † Thomas N. Chiesl, † Russell D. Haynes, ‡ Jong-In Won, †,| and Annelise E. Barron* ,†,‡ Department of Chemical and Biological Engineering and Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208 We report a new, bioconjugate approach to performing highly multiplexed single-base extension (SBE) assays, which we demonstrate by genotyping a large panel of point mutants in exons 5-9 of the p53 gene. A series of monodisperse polyamide “drag-tags” was created using both chemical and biological synthesis and used to achieve the high-resolution separation of genotyping reac- tion products by microchannel electrophoresis without a polymeric sieving matrix. A highly multiplexed SBE reac- tion was performed in which 16 unique drag-tagged primers simultaneously probe 16 p53 gene loci, with an abbreviated thermal cycling protocol of only 9 min. The drag-tagged SBE products were rapidly separated by free- solution conjugate electrophoresis (FSCE) in both capil- laries and microfluidic chips with genotyping accuracy in excess of 96%. The separation requires less than 70 s in a glass microfluidic chip, or about 20 min in a commercial capillary array sequencing instrument. Compared to gel electrophoresis, FSCE offers greater freedom in the design of SBE primers by essentially decoupling the length of the primer and the electrophoretic mobility of the genotyping products. FSCE also presents new possibilities for the facile implementation of SBE on integrated microfluidic electrophoresis devices for rapid, high-throughput genetic mutation detection or SNP scoring. Although the sequencing of the first human genome was completed amidst much fanfare in 2003, a great need still exists for studying variability among different individual human genomes as well as among the genomes of other organisms. More than 90% of the genetic variability among humans is thought to consist of single-nucleotide polymorphisms (SNPs), and efforts are ongoing to map more than 300 000 SNPs. 1,2 While many SNPs have no significant impact on protein expression or cell function, specific SNPs have been found to predispose individuals to certain diseases, including sickle cell anemia and Alzheimer’s disease. 3,4 For example, mutations in the p53 gene have been implicated in a wide variety of human cancers, with missense mutations comprising a large majority of deleterious p53 sequence alterations. 5-9 Furthermore, sequence polymorphisms in a variety of interacting genes are suspected to be responsible for complex diseases such as cancer, heart disease, and psychiatric disorders; the results of multiplexed, multigene SNP analyses in large populations are expected to enable valuable insights into such conditions. 1,10 A wide variety of techniques have been proposed for SNP detection, and many of these methods have recently been reviewed. 11,12 Most methods begin with PCR amplification of the gene region to be tested, typically followed by an enzymatic allele discrimination reaction, and then the detection and identification of the reaction products. Biomolecule detection schemes based on fluorescence or fluorescence resonance energy transfer, mass spectrometry, or microarrays can allow accurate identification of allele-specific products. Each method has its advantages and disadvantages with respect to simplicity, sensitivity, ease of multiplexing, throughput, and cost; the choice of SNP genotyping method varies, depending on the specific needs and resources of each laboratory. One widely used technique for allele discrimination based on the synthesis activity of DNA polymerase is the single-base extension (SBE) assay, also known as mini-sequencing or primer- * Corresponding author. Phone: (847) 491-2778. Fax: (847) 491-3728. E-mail: a-barron@northwestern.edu. † Department of Chemical and Biological Engineering. ‡ Department of Chemistry. § Present address: Sandia National Laboratories, Livermore, CA. | Present address: Department of Chemical Engineering, Hongik University, Seoul, South Korea. (1) Collins, F. S.; Brooks, L. D.; Chakravarti, A. Genome Res. 1998, 8, 1229- 1231. (2) Brookes, A. J. Gene 1999, 234, 177-186. (3) Strittmatter, W. J.; Saunders, A. M.; Schmechel, D.; Pericakvance, M.; Enghild, J.; Salvesen, G. S.; Roses, A. D. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 1977-1981. (4) Strittmatter, W. J.; Roses, A. D. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 4725-4727. (5) Greenblatt, M. S.; Bennett, W. P.; Hollstein, M.; Harris, C. C. Cancer Res. 1994, 54, 4855-4878. (6) Soussi, T.; Beroud, C. Nat. Rev. Cancer 2001, 1, 233-240. (7) Soussi, T.; Lozano, G. Biochem. Biophys. Res. Commun. 2005, 331, 834- 842. (8) Birch, J. M.; Alston, R. D.; McNally, R. J.; Evans, D. G.; Kelsey, A. M.; Harris, M.; Eden, O. B.; Varley, J. M. Oncogene 2001, 20, 4621-4628. (9) Olivier, M.; Goldgar, D. E.; Sodha, N.; Ohgaki, H.; Kleihues, P.; Hainaut, P.; Eeles, R. A. Cancer Res. 2003, 63, 6643-6650. (10) Kirk, B. W.; Feinsod, M.; Favis, R.; Kliman, R. M.; Barany, F. Nucleic Acids Res. 2002, 30, 3295-3311. (11) Landegren, U.; Nilsson, M.; Kwok, P. Y. Genome Res. 1998, 8, 769-776. (12) Chen, X.; Sullivan, P. F. Pharmacogenomics J. 2003, 3, 77-96. Anal. Chem. 2007, 79, 1848-1854 1848 Analytical Chemistry, Vol. 79, No. 5, March 1, 2007 10.1021/ac061903z CCC: $37.00 © 2007 American Chemical Society Published on Web 01/26/2007