title: Real-time PCR to study the sequence specific magnetic purification of DNA.
abstract: 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. However, 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 efficiency 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.
authors.affiliation: IMEC, SSET/FNS, B-3001 Leuven, Belgium. sara.peeters@imec.be
authors.forename: Sara
authors.initials: S
authors.lastname: Peeters
authors.forename: Tim
authors.initials: T
authors.lastname: Stakenborg
authors.forename: Frederik
authors.initials: F
authors.lastname: Colle
authors.forename: ChengXun
authors.initials: C
authors.lastname: Liu
authors.forename: Liesbet
authors.initials: L
authors.lastname: Lagae
authors.forename: Marc
authors.initials: M
authors.lastname: Van Ranst
mesh_terms: D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D052162:Human papillomavirus 16 / Q000235:genetics; D008280:Magnetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019809:Streptavidin / Q000737:chemistry
chemical_list: D004279:DNA, Viral; D019809:Streptavidin