(Pcr) for Dna Analysis M

ON-CHIP INTEGRATION OF SAMPLE PRE- TREATMENT AND MULTIPLEX POLYMERASE CHAIN REACTION (PCR) FOR DNA ANALYSIS M. Brivio1, D. Snakenborg1, E. Søgaard1, A. Ahlford2, A.C. Syvänen2, J. P. Kutter1 and A. Wolff1 1DTU Nanotech, Technical University of Denmark, DENMARK 2Molecular Medicine, University Hospital, Uppsala, SWEDEN ABSTRACT In this paper we present a modular lab-on-a-chip system for integrated sample pre-treatment (PT) by magnetophoresis and DNA amplification by polymerase chain reaction (PCR). It consists of a polymer-based microfluidic chip mounted on a cus- tom-made thermocycler (Figure 1) and includes a simple and efficient method for switching the liquid flow between the PT and PCR chamber. Purification of human genomic DNA from EDTA-treated blood and multiplex PCR were successfully carried out on-chip using the developed lab-on-a-chip system. KEYWORDS: lab-on-a-chip, integration, multiplex PCR, diagnostics INTRODUCTION PCR is widely used for DNA amplification and subsequent analysis for many applications in medical diagnostics, forensics, recombinant technology and molecular genetics. A pre-treatment step is often required in order to remove PCR inhibitors from the sample and to pre-concentrate DNA, prior to its amplification by PCR. Over the past fifteen years a number of PCR chips have been proposed, which exploit the advantages of miniaturization, such as fast mass and heat transfer, low reagents consumption, and portability. However, the integration of sample pre- treatment and PCR on the same microfluidic platform is still a challenge [1]. In this work, a lab-on-a-chip device for integrated human genomic DNA purification Figure 1. Photograph of the lab-on-a- and multiplex PCR was developed and chip device for integrated DNA purifi- successfully tested for the amplification of cation and PCR: (a) automated rota- thirteen DNA fragments. This multiplex tion is provided by a stepper motor; (b) PCR was developed as the first step of an a peltier element for thermocycling assay for genotyping of point mutations and an array of magnets for capturing and single nucleotide polymorphisms magnetic beads are placed in a chip (SNPs) [2]. These include five mutations holder below the biochip. in three codons (175, 248 and 273) of the Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA 978-0-9798064-1-4/µTAS2008/$20©2008CBMS 1737 p53 gene, which are the most frequent known mutations in common cancers. EXPERIMENTAL The microfluidic biochip consists of a stator, with channels (200 µm wide and 200 µm deep) and chambers (14 mm long, 4 mm wide and 200 µm deep), and a stepper motor-controlled rotor containing connection channels (Figure 1). O-rings provide sufficient sealing during rotation and PCR thermocycling. The biochip is mounted on a polycarbonate holder, which accommodates an array of permanent magnets and a Peltier element below the PT and PCR chamber, respectively (Figure 1). Rotation allows easy switching between two positions, thereby connecting the PT chamber either to the outlet or to the PCR chamber (Figure 2). Microfluidic structures were fabricated in poly(methyl methacrylate) substrates by micromilling. Figure 2. Schematic representation of the different positions of the fluidic network of the biochip: (a) PT chamber connected to outlet 1 (waste); (b) PT connected to PCR chambers allowing to collect the products at outlet 2. RESULTS AND DISCUSSION The lab-on-a-chip system was successfully used to purify human genomic DNA from EDTA-treated blood samples. Sample pre-treatment was done by magnetophoresis, using the MagneSil®KF Genomic System [3]. Blood samples pre-mixed with magnetic beads and lysis buffer were injected into the PT chamber. DNA molecules released upon cell lysis bind to the surface of the beads, which are captured by an external magnet at the bottom of the chamber. During sample injection and washing the PT chamber was connected to outlet 1 (Figure 2a). After washing, the position of the rotor was switched to connect the PT to the PCR chamber (Figure 2b) and PCR- ready DNA was eluted from the beads. Figure 3 shows the products of multiplex PCR carried out off-chip using on-chip purified DNA as a template. Freeze-drying was used to store PCR reagents [4] in the PCR chamber of the biochip, allowing the integration of sample pre-treatment and PCR without adding complexity to the fluidic network. As shown in Figure 4, multiplex PCR was successfully carried out in the lab-on-a-chip system by dissolving freeze-dried reagents with eluted DNA. CONCLUSIONS We believe that the lab-on-a-chip system presented in this abstract is a powerful tool for integrating DNA purification and DNA amplification by PCR. By modify- ing the fluidic network layout and the reagents stored in the chambers this set-up may be adapted to various applications. Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA 1738 Figure 3. (a) Electropherogram and (b) gel representation of the products of a multiplex PCR carried out off-chip using human genomic DNA as a template. The template was purified on the lab-on-a-chip set-up from EDTA-treated whole blood. Figure 4. (a) Electropherogram and (b) gel representation of the products of a multiplex PCR carried out on the lab-on-a-chip set-up. 7 µL of genomic DNA solution were added to freeze-dried PCR reagents stored on-chip ACKNOWLEDGEMENTS The authors would like to thank the Klinisk Immunologisk Center at Gentofte Hospital for kindly providing the blood samples and both the EC SMART- BioMEMS project (IST-016554) and the Swedish Research Council for Science and Technology for financial support. REFERENCES [1] C. Zhang and D. Xing, Miniaturized PCR chips for nucleic acid amplification and analysis: latest advances and future trends, Nucleic Acids Res., 35, 4223 (2007). [2] K. Lindroos, S. Sigurdsson, K. Johansson, L. Ronnblom, AC. Syvänen, Multi- plex SNP genotyping in pooled DNA samples by a four-colour microarray system, Nucleic Acids Res., 30, e70 (2002). [3] http://www.promega.com/tbs/tb322/tb322.pdf [4] M. Brivio, Y. Li, A. Ahlford, B.G. Kjeldsen J.L. Reimers M. Bu AC. Syvänen, D.D. Bang, A. Wolff, A simple and efficient method for on-chip storage of freeze-dried reagents: towards lab-on-a-chip systems for point-of-care DNA diagnostics, Proc. Micro Total Analysis Systems 2007, 1, 59 (2007). Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences October 12 - 16, 2008, San Diego, California, USA 1739.

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