The Evaluation of Magnetic Polymethacrylate-based Microspheres Used for Solid Phase DNA Micro-Extraction

Štěpánka Trachtová,Hana Zapletalová,Bohuslav Rittich,Alena Španová,Daniel Horák,Hana Kolářová

doi:10.3390/chromatography2020156

Abstract

Using magnetic particles as a solid-phase extraction system is the most frequently used micro-technique for DNA isolation. Particles with a complete covering of magnetic cores by a polymer are hence preferred. Quantitative polymerase chain reaction (qPCR) was used for the evaluation of the polymer coating efficiency of hydrophilic magnetic 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 was identified by the shift in Cq values (ΔCq) after the addition of different amounts of microspheres to PCR mixtures. With the increase of microsphere concentrations, the shift in Cq values to higher values was usually observed. P(HEMA-co-GMA) microspheres containing carboxyl groups extinguished the fluorescence at concentrations over 2 mg mL−1 in a PCR mixture without any influence on the synthesis of PCR products. No PCR products (inhibition of DNA amplification) were detected in the presence of more than 0.8 mg mL−1 in the PCR mixture of PGMA microspheres. Atomic force microscopy (AFM) was used for the determination of the surface morphology of the microspheres. The microspheres were spherical, and their surface was non-porous.

Highlights

Amplification of nucleic acids using polymerase chain reaction (PCR) has become a powerful diagnostic tool for the identification of microorganisms in foods [1,2,3,4], soil communities [5], clinical samples [6], forensic analysis [7], etc
The presence of PCR inhibitors in DNA samples causes a number of problems, ranging from reduced amplification efficiency and assay sensitivity to complete reaction failure
Numerous solid phase systems involving different micro- and nano-sized magnetic carriers and appropriate buffer systems have been used for DNA isolation [12,13]

Summary

Introduction

Amplification of nucleic acids using polymerase chain reaction (PCR) has become a powerful diagnostic tool for the identification of microorganisms in foods [1,2,3,4], soil communities [5], clinical samples [6], forensic analysis [7], etc. There is no problem with the amplification of DNA isolated from pure bacterial cultures. The presence of PCR inhibitors (of unknown amounts and compositions) in DNA samples causes a number of problems, ranging from reduced amplification efficiency and assay sensitivity to complete reaction failure (false-negative results). The effects of the inhibitors can be reduced by the selection of an appropriate nucleic acid isolation method. The presence of interfering compounds released from magnetic particles decreased the PCR sensitivity or led to false-negative results [14]

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Results