Sensitive on-chip quantitative real-time PCR performed on an adaptable and robust platform.
A robust, flexible and efficient system for performing high sensitivity quantitative on-chip real-time PCR for research purposes is presented. The chips used consist of microchannels etched in silicon. The surface in the channels is a thermally grown silicon dioxide and the channel is sealed by a glass lid. The chips contain four PCR chambers but this number can be increased for further multiplexing. Contrary to PCR chips with oil covered open chambers, these channel-like chambers are easily integrated in lab-on-a-chip devices. The temperature is controlled by a Peltier element and the fluorochrome detector system is a commercially available fluorescence stereo microscope equipped with a CCD camera. The setup shows an excellent signal-to-noise ratio of about 400 compared to that of about 150 obtained in a commercial real time PCR machine. A detection limit of a few copies of target molecules is found, which is 100 to 100,000-fold better than other on-chip real-time PCR systems presented in the literature. This demonstrates that the PCR system can be used for critical applications. We also demonstrate that high quality melting curves can be obtained. Such curves are important in lab-on-a-chip systems for identification of amplified product. The usability of the system is validated by performing quantitative on-chip measurements of the amount of specific gene sequences co-immunoprecipitated with various posttranslationally modified histone proteins. Similar results are obtained from on-chip experiments and experiments carried out in a commercial system on larger sample volumes.
Lund-Olesen
Torsten
T
DTU Nanotech-Department of Micro and Nanotechnology, Technical University of Denmark, Building 345 East, 2800, Kongens Lyngby, Denmark.
Dufva
Martin
M
DTU Nanotech-Department of Micro and Nanotechnology, Technical University of Denmark, Building 345 East, 2800, Kongens Lyngby, Denmark.
Dahl
John Arne
JA
Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, P.O. Box 1112, Blindern, 0317, Oslo, Norway.
Collas
Philippe
P
Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, P.O. Box 1112, Blindern, 0317, Oslo, Norway.
Hansen
Mikkel Fougt
MF
DTU Nanotech-Department of Micro and Nanotechnology, Technical University of Denmark, Building 345 East, 2800, Kongens Lyngby, Denmark. mfh@mic.dtu.dk.
D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000032:analysis*; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D012680:Sensitivity and Specificity
D004273:DNA, Neoplasm