MICROFLUIDIC APP FEATURING NESTED PCR FOR FORENSIC SCREENING ASSAY ON OFF-THE-SHELF THERMOCYCLER M. Keller 1,2* , J. Naue 3,4 , P. Papireddy Vinayaka 1,2 , O. Strohmeier 2 , D. Mark 2 , U. Schmidt 3 , R. Zengerle 1,2,5 and F. von Stetten 1,2 1 Laboratory for MEMS Applications, IMTEK – Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, GERMANY; 2 HSG-IMIT – Institut für Mikro- und Informationstechnik, Georges-Koehler-Allee 103, 79110 Freiburg, GERMANY; 3 Institute of Legal Medicine, Freiburg University Medical Center, Albertstr. 9, 79104 Freiburg, GERMANY; 4 Faculty of Biology, University of Freiburg, Schaenzlestr. 1, 79104 Freiburg, GERMANY and 5 BIOSS – Centre for Biological Signalling Studies, Schaenzlestr. 18, University of Freiburg, 79110 Freiburg, GERMANY ABSTRACT Microfluidic Apps are a novel class of microfluidic chips designed to inbuilt process automation into standard laboratory instruments [1]. This approach circumvents costly robotic liquid handling systems. We demonstrate a new Microfluidic App for automation of forensic screening assays using an off-the-shelf centrifugal real-time thermocycler. This instrument is used for nucleic acid testing by polymerase chain reaction (PCR). Compared to previous approaches, which enabled centrifugal microfluidic liquid handling by control of rotational frequency [2] our novel App excels by implementation of a multistep liquid handling protocol at constant rotational frequency using centrifugo- thermopneumatic actuation [3]. Automation of the forensic assay integrates the following steps into a microfluidic LabDisk segment referred to as GeneSlice: PCR pre-amplification of the DNA-sample and a no template-control, followed by aliquoting of the pre-amplified DNA into 15 reaction wells (5.1 ± 0.4 µl, CV 6.9 %, N = 4), in which amplification by nested PCR including internal positive- and extraction-controls with subsequent melting curve analysis is performed. Discrimination of human-, dog-, and red deer-DNA could be successfully demonstrated. The novel Microfluidic App enables process automation at low cost and avoids the risk of cross-contaminations typically found in non-integrated nested PCR analyses. KEYWORDS: Microfluidic App, centrifugal microfluidics, forensics, nucleic acid testing, real-time PCR, thermocycler INTRODUCTION Forensic DNA samples often contain low and/or limited amounts of DNA, which is challenging when a variety of targets have to be detected, especially if multiplexing is not possible. Regarding species determination, nested PCR can solve this problem by combining a universal pre-amplification (PA) prior to specific main-amplification (MA) [4]. Multiple required manual handling steps make nested PCR in forensics susceptible to contamination. A self-contained microfluidic integration of nested PCR steps highly reduces this risk. For the first time, we present a suitable automation as Microfluidic App that can be operated on an unmodified thermocycler (Rotor-Gene Q, QIAGEN GmbH, Hilden, Germany) enabling implementation into any laboratory (Figure 1) [2]. We have greatly expanded a proof-of-concept [3] to actuate fluids at the low (400 rpm) and constant rotational frequency of the Rotor-Gene using centrifugo-thermopneumatic unit operations. Figure 1: Microfluidic Apps for nested PCR feature low contamination risk in a self-contained Lab-on-a-Chip system, which can be processed in an unmodified standard laboratory real-time PCR thermocycler (Rotor-Gene Q, Qiagen, Germany). A special rotor holder enables insertion of four GeneSlices per run. 978-0-9798064-6-9/µTAS 2013/$20©13CBMS-0001 320 17th International Conference on Miniaturized Systems for Chemistry and Life Sciences 27-31 October 2013, Freiburg, Germany