Real-Time PCR to Study the Sequence Specific Magnetic Purification of DNA Sara Peeters IMEC, SSET/FNS, B-3001 Leuven, Belgium KULeuven, Department of Microbiology and Immunology, B-3001 Leuven, Belgium Tim Stakenborg IMEC, SSET/FNS, B-3001 Leuven, Belgium Frederik Colle IMEC, SSET/FNS, B-3001 Leuven, Belgium University of Antwerp, Department of Biomedical Sciences, B-2610 Antwerp, Belgium Applied Molecular Genomics Group, Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Belgium ChengXun Liu IMEC, SSET/FNS, B-3001 Leuven, Belgium Liesbet Lagae IMEC, SSET/FNS, B-3001 Leuven, Belgium KULeuven, Department of Physics and Astronomy, B-3001 Leuven, Belgium Marc Van Ranst KULeuven, Department of Microbiology and Immunology, B-3001 Leuven, Belgium DOI 10.1002/btpr.492 Published online September 27, 2010 in Wiley Online Library (wileyonlinelibrary.com). The performance of various molecular techniques using complex biological samples greatly depends on the efficient separation and purification of DNA targets. In recent years, magnetic separation technology making use of small magnetic beads, has gained immense popularity. Most of these methods rely on the non-specific adsorption of DNA/RNA. How- ever, as presented here, when functionalizing the beads with complementary DNA probes, the target of interest can selectively be isolated. Such sequence specific purification was evaluated for short DNA targets by means of simple fluorescent measurements, resulting in purification efficiencies around 80%. Besides standard fluorescent techniques, a real-time PCR (qPCR) method was applied for monitoring the purification of longer DNA targets. This qPCR method was specifically optimized for directly quantifying the purification effi- ciency of low concentrated DNA targets bound to magnetic beads. Additionally, parameters possibly affecting the magnetic isolation, including the length of the used capture probe or the hybridization location, were investigated. Using optimized conditions in combination with qPCR, purification efficiencies between 60% and 80% were observed and this over a large concentration window. These data also show the power of a direct qPCR approach to monitor the magnetic isolation of DNA at very low concentrations. V V C 2010 American Insti- tute of Chemical Engineers Biotechnol. Prog., 26: 1678–1684, 2010 Keywords: selective DNA isolation, on-bead PCR, magnetic particles, DNA, quantification, sample preparation Introduction In many biomedical applications it is required to specifically isolate, enrich, and purify biological analytes from clinical or environmental samples. 1 Despite the importance of sample preparation it is still considered as the most time-consuming and, irreproducible step within biochemical analysis. 2,3 Mag- netic beads have already proven their great potential to fasten, simplify, and standardize current purification protocols. 4,5 As such, they are preferentially being used in more automated sample preparation procedures or, with further down-scaling and integration, in the development of portable or lab-on-a- chip systems. 5–10 Most commercially available magnetic sepa- ration systems isolate all DNA from a sample by means of sim- ple adsorption. 6,11,12 However, for some applications a Correspondence concerning this article should be addressed to S. Peeters at sara.peeters@imec.be. 1678 V V C 2010 American Institute of Chemical Engineers