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Solid-Phase Reversible Immobilization in Microfluidic Chips for the Purification of Dye-Labeled DNA Sequencing Fragments Yichuan Xu, Bikas Vaidya, Ami B. Patel,† Sean M. Ford,‡ Robin L. McCarley, and Steven A. Soper*
Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803-1804
In this manuscript, we discuss the use of photoactivated polycarbonate (PC) for purification of dye-labeled terminator sequencing fragments using solid-phase reversible immobilization (SPRI) prior to gel electrophoretic sorting of these DNAs. An immobilization bed for the DNA purification was produced by exposing a posted microchannel to UV radiation, which induced a surface photooxidation reaction, resulting in the production of carboxylate groups. The immobilization microchannel contained microposts to increase the loading level of DNAs to improve signal intensity without the need for preconcentration. By suspending the sequencing cocktail in an immobilization buffer (TEG/ethanol), the DNA fragments demonstrated a high affinity for this carboxylated surface. The loading density of DNAs to this activated surface was found to be 3.9 pmol cm-2. The captured DNA could be subsequently released from the surface by incubation with ddH2O. SPRI cleanup of dye-terminator sequencing fragments using the photoactivated PC chip and slab gel electrophoresis produced a read length comparable to the conventional SPRI format, which utilized carboxylated magnetic beads and a magnetic field. The read length for the PC-SPRI format was found to be 620 bases with a calling accuracy of 98.9%. The PC-SPRI cleanup format was also integrated to a capillary gel electrophoresis (CGE) system. The PC-SPRI method was shown to effectively remove excess dye terminator from the CGE tract, but yielded lower plate numbers, as compared to a direct injection method with purification accomplished offchip. The loss in efficiency was found to result primarily from the extended injection time associated with the microchip purification method. The construction of the first rough draft of the human genome sequence by both the private and government/academic sectors has been reported.1,2 Although the human genome has been successfully sequenced, there continues to be a need toward reducing costs, increasing automation, and simplifying technology so that sequencing in areas such as functional genomics, sequenc* To whom correspondence should be addressed. † Current address: Department of Chemistry, Centenary College, Shreveport, LA 70811. ‡ Current address: Diagnostics Product Corporation, 5700 W. 96th St., Los Angeles, CA 90045. (1) Venter, J. C.; et al. Science 2001, 291, 1304-1351. (2) Lander, E. S.; et al. Nature 2001, 409, 860-921. 10.1021/ac030031n CCC: $25.00 Published on Web 05/22/2003
© 2003 American Chemical Society
ing genomes of model organisms, comparative and population genomics, de novo sequencing, and the detection of single nucleotide polymorphisms (SNPs) can become more practical. In addition, improvements in sequencing technology can potentially move large genomic projects into smaller laboratories rather than require that they be restricted to major genome sequencing factories. In the past decade, extensive research has been dedicated to developing miniaturized separation techniques to reduce the development time and improve automation of the electrophoretic process, an example being capillary gel electrophoresis (CGE)3-10 and, recently, capillary array electrophoresis (CAE).11-16 Electrophoresis on microchips is an emerging new technology that promises to lead the next revolution in DNA sequencing. Microelectrophoresis platforms have already been shown to separate DNA restriction fragments,17,18 PCR products,19 short oligonucleotides,20 and short tandem repeats21 rapidly and with reasonable resolution. High-speed separations of DNA sequencing fragments have also been achieved using these chips.22,23 Recently, (3) Dovichi, N. J.; Zhang, J. Z. Angew. Chem.-Int. Ed. 2000, 39, 4463-4469. (4) Dovichi, N. J. Electrophoresis 1997, 18, 2393-2399. (5) Dovichi, N. J. Hum. Mutat. 1993, 2, 82-84. (6) Quesada, M. A.; Rye, H. S.; Gingrich, J. C.; Glazer, A. N.; Mathies, R. A. Biotechniques 1991, 10, 616-625. (7) Rye, H. S.; Quesada, M. A.; Peck, K.; Mathies, R. A.; Glazer, A. N. Nucleic Acids Res. 1991, 19, 327-333. (8) Drossman, H.; Luckey, J. A.; Kostichka, A. J.; Dcunha, J.; Smith, L. M. Anal. Chem. 1990, 62, 900-903. (9) Luckey, J. A.; Drossman, H.; Kostichka, A. J.; Mead, D. A.; Dcunha, J.; Norris, T. B.; Smith, L. M. Nucleic Acids Res. 1990, 18, 4417-4421. (10) Luckey, J. A.; Drossman, H.; Kostichka, T.; Smith, L. M. Methods Enzymol. 1993, 218, 154-172. (11) Huang, X. H. C.; Quesada, M. A.; Mathies, R. A. Anal. Chem. 1992, 64, 2149-2154. (12) Mathies, R. A.; Huang, X. C. Nature 1992, 359, 167-169. (13) Huang, X. H. C.; Quesada, M. A.; Mathies, R. A. Anal. Chem. 1992, 64, 967-972. (14) Zhang, J. Z.; et al. Nucleic Acids Res. 1999, 27, e36. (15) Zhang, J.; Yang, M.; Puyang, X.; Fang, Y.; Cook, L. M.; Dovichi, N. J. Anal. Chem. 2001, 73, 1234-1239. (16) Anazawa, T.; Takahashi, S.; Kambara, H. Anal. Chem. 1996, 68, 26992704. (17) Woolley, A. T.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1134811352. (18) Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1996, 68, 720-723. (19) Waters, L. C.; Jacobson, S. C.; Kroutchinina, N.; Khandurina, J.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1998, 70, 5172-5176. (20) Effenhauser, C. S.; Paulus, A.; Manz, A.; Widmer, H. M. Anal. Chem. 1994, 66, 2949-2953. (21) Schmalzing, D.; Koutny, L.; Adourian, A.; Belgrader, P.; Matsudaira, P.; Ehrlich, D. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10273-10278. (22) Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676-3680.
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microfabricated capillary array electrophoresis (µCAE) chips have also been fabricated to further demonstrate increased throughput using these devices.24-26 Although microchips may become the platform of choice in most sequencing applications using electrophoresis as the final step in the process, it is clear that challenges in data quality will continue to be an important issue when using these devices. For example, the short length of the separation channel limits the effective read length obtainable using these devices.23 Proper preparation of the sequencing ladders prior to electrophoresis will be an integral step in using these devices, since the quality of the read depends intimately on the quality of the sample input into the device, especially for sequencing applications. In 1998, RuizMartinez et al. and Salas-Solano et al. carried out critical investigations on the effects of reaction matrix components on read length using capillary gel electrophoresis for the separation.27-29 Desalting and template removal were found to be essential for maintaining read lengths >500 bases. Purification of sequencing reaction products in these studies was predominantly accomplished by conventional methods, such as acetate/ethanol precipitation,30 phenol/chloroform extraction,31 or gel filtration in spin columns or microtiter plate formats.32-34 Unfortunately, these methods require large sample volumes and high-speed centrifugation, which may not be practical for integration into miniaturized sequencing platforms. There have been examples that discuss fully automated systems using capillaries and microfluidics to process sequencing samples and send the processed samples to a capillary gel column for electrophoretic sorting.35-38 Electroosmotically driven flow, dyeterminator chemistry, and cycle sequencing was used to sequence pGEM templates in a capillary nanoreactor with the cycle sequencing products purified (remove excess salts and dyeddNTPs) using free solution capillary zone electrophoresis prior to loading onto the gel column.38 Paegel and co-workers recently implemented a gel immobilization strategy in a microfluidic chip in which appropriately prepared PCR products and the associated (23) Schmalzing, D.; Adourian, A.; Koutny, L.; Ziaugra, L.; Matsudaira, P.; Ehrlich, D. Anal. Chem. 1998, 70, 2303-2310. (24) Schmalzing, D.; Tsao, N.; Koutny, L.; Chisholm, D.; Srivastava, A.; Adourian, A.; Linton, L.; McEwan, P.; Matsudaira, P.; Ehrlich, D. Genome Res. 1999, 9, 853-858. (25) Paegel, B. M.; Hutt, L. D.; Simpson, P. C.; Mathies, R. A. Anal. Chem. 2000, 72, 3030-3037. (26) Liu, S. R.; Shi, Y. N.; Ja, W. W.; Mathies, R. A. Anal. Chem. 1999, 71, 566573. (27) Salas-Solano, O.; Carrilho, E.; Kotler, L.; Miller, A. W.; Goetzinger, W.; Sosic, Z.; Karger, B. L. Anal. Chem. 1998, 70, 3996-4003. (28) Salas-Solano, O.; Ruiz-Martinez, M. C.; Carrilho, E.; Kotler, L.; Karger, B. L. Anal. Chem. 1998, 70, 1528-1535. (29) Ruiz-Martinez, M. C.; Salas-Solano, O.; Carrilho, E.; Kotler, L.; Karger, B. L. Anal. Chem. 1998, 70, 1516-1527. (30) Sambrok, J.; Fritsh, E. F.; Maniates, T. Molecular Cloning - A Laboratory Manuel; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989. (31) Figeys, D.; Ahmadzedeh, H.; Arriaga, E.; Dovichi, N. J. J. Chromatogr., A 1996, 744, 325-331. (32) Hilderman, D.; Muller, D. BioTechniques 1997, 22, 878. (33) Devaney, J.; Marino, M.; Williams, P.; Weaver, K.; Truner, K.; Belgrader, P. Appl. Theor. Electrophor. 1996, 6, 11-17. (34) Wang, K.; Ban, L.; Boysen, C.; Hood, L. Anal. Biochem. 1995, 226, 85-91. (35) Swerdlow, H.; Jones, B. J.; Wittwer, C. T. Anal. Chem. 1997, 69, 848-855. (36) Tan, H. D.; Yeung, E. S. Anal. Chem. 1997, 69, 664-674. (37) He, Y.; Zhang, Y. H. H.; Yeung, E. S. J. Chromatogr., A 2001, 924, 271284. (38) He, Y.; Pang, H. M.; Yeung, E. S. J. Chromatogr., A 2000, 894, 179-190.
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microfluidic circuit were used for the purification of the DNA sequencing samples.39 The PCR primers contained sequences complementary to short capture probes tethered covalently to a gel matrix. Because of hybridization between the PCR-generated sequencing products and the tethered capture probes, the authors were able to purify the templates in
