title: Real time quantitative amplification detection on a microarray: towards high multiplex quantitative PCR.
abstract: Quantitative real-time polymerase chain reaction (qrtPCR) is widely used as a research and diagnostic tool. Notwithstanding its many powerful features, the method is limited in the degree of multiplexing to about 6 due to spectral overlap of the available fluorophores. A new method is presented that allows quantitative amplification detection at higher multiplexing by the integration of amplification in solution and monitoring via hybridization to a microarray in real-time. This method does not require any manipulation of the PCR product and runs in a single closed chamber. Employing labeled primers, one of the main challenges is to measure surface signals against a high fluorescence background from solution. A compact, confocal scanner is employed, based on miniaturized optics from DVD technology and combined with a flat thermocycler for simultaneous scanning and heating. The feasibility of this method is demonstrated in singleplex with an analytical sensitivity comparable to routine qrtPCR.
authors.affiliation: Philips Research, High Tech Campus 11, 5656 AE Eindhoven, The Netherlands. anke.pierik@philips.com
authors.forename: Anke
authors.initials: A
authors.lastname: Pierik
authors.forename: Marius
authors.initials: M
authors.lastname: Boamfa
authors.forename: Martijn
authors.initials: M
authors.lastname: van Zelst
authors.forename: Danielle
authors.initials: D
authors.lastname: Clout
authors.forename: Henk
authors.initials: H
authors.lastname: Stapert
authors.forename: Frits
authors.initials: F
authors.lastname: Dijksman
authors.forename: Dirk
authors.initials: D
authors.lastname: Broer
authors.forename: Reinhold
authors.initials: R
authors.lastname: Wimberger-Friedl
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D017931:DNA Primers; D004867:Equipment Design; D005456:Fluorescent Dyes; D008957:Models, Genetic; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation* / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
chemical_list: D001426:Bacterial Proteins; D017931:DNA Primers; C096686:FemA protein, Bacteria; D005456:Fluorescent Dyes