title: Real-Time Polymerase Chain Reaction as a Tool for Evaluation of Magnetic Poly(Glycidyl methacrylate)-Based Microspheres in Molecular Diagnostics.
abstract: DNA amplification by real-time polymerase chain reaction (RT-PCR) was used for the evaluation of efficiency of polymer coating of magnetic hydrophilic poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) and poly(glycidyl methacrylate) (PGMA) microspheres with/without carboxyl groups. The inhibition effect of magnetic microspheres on real-time polymerase chain reaction (RT-PCR) course was evaluated by regression analysis after the addition of different concentrations of tested microspheres to PCR mixtures. Microspheres mostly did not interfere in RT-PCR till the concentration 50 µg/25 µl PCR mixture. No relationship between Fe content (and microsphere diameter) and inhibition effect was found. Microspheres containing carboxyl groups extinguished the fluorescence at lower concentrations (10-20 µg/25 µl PCR mixture) without inhibition of DNA amplification as PCR products were detected using agarose gel electrophoresis. Negative effect of maghemite on PCR course was partially reduced by coating of magnetic core by silica or polymers. Two inhibition mechanisms of DNA amplification were discussed in this work.
authors.forename: Stepánka
authors.initials: S
authors.lastname: Trachtová
authors.forename: Alena
authors.initials: A
authors.lastname: Spanová
authors.forename: Daniel
authors.initials: D
authors.lastname: Horák
authors.forename: Hana
authors.initials: H
authors.lastname: Kozáková
authors.affiliation: Brno University of Technology, Faculty of Chemistry, Purkynova 118, CZ-612 00 Brno, Czech Republic. rittich@fch.vutbr.cz.
authors.forename: Bohuslav
authors.initials: B
authors.lastname: Rittich
mesh_terms: D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D008863:Microspheres; D057089:Pathology, Molecular / Q000379:methods*; D011109:Polymethacrylic Acids / Q000737:chemistry*; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004269:DNA, Bacterial; D058185:Magnetite Nanoparticles; D011109:Polymethacrylic Acids; C042535:polyglycidyl methacrylate