Real-Time Polymerase Chain Reaction as a Tool for Evaluation of Magnetic Poly(Glycidyl methacrylate)-Based Microspheres in Molecular Diagnostics

Author(s): Stepanka Trachtova, Alena  Spanova, Daniel Horak, Hana Kozakova, Bohuslav Rittich.

Journal Name: Current Pharmaceutical Design

Volume 22 , Issue 5 , 2016

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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/25µl 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/25µl 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.

Keywords: Magnetic microspheres, inhibitory effect, real-time polymerase chain reaction, regression analysis.

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Article Details

VOLUME: 22
ISSUE: 5
Year: 2016
Page: [639 - 646]
Pages: 8
DOI: 10.2174/1381612822666151228105006
Price: $58

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