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Reversible immobilisation of DNA on newly designed magnetic carriers
Kubisz, Petr ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The aim of work was an optimization of separation deoxyribonucleic acid (DNA) with the use of nucleic acid reversible adsorption to the surface of magnetic particles coated by functional groups. Six carriers were verificated for DNA isolation: P (HEMA-co-GMA) ox, F-kol B 30 ox, F-kol 77 ox, F-kol B100 ox, F-kol 135 ox, coated with carboxyl groups and Perovskit 439 (coated by silicone). Bacterial DNA was isolated by phenol extraction procedure, first. DNA was reversibly bond to magnetis carrier in the presence of high concentration of NaCl ( 5 M) and poly (ethylene glycol) (PEG 6000). The final PEG and NaCl concentrations of 16.0 % (w/v) and 2.0 M, respectively, were used.DNA was eluted into TE buffer. The quality of extracted DNA was checked by PCR amplification. It was found out that although different quantities of DNA were isolated, the quality of isolated DNA was always compatible with PCR. Nanoparticles Perovskit 439 had the best separative characteristics in comparison to the other magnetic carriers because highest amounts of DNA was isolated. However, next optimisation of DNA separation procedure is required for the use of studied microspheres in real samples.
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Reversible immobilisation of DNA on newly designed magnetic carriers
Kubisz, Petr ; Španová, Alena (referee) ; Rittich, Bohuslav (advisor)
The aim of work was an optimization of separation deoxyribonucleic acid (DNA) with the use of nucleic acid reversible adsorption to the surface of magnetic particles coated by functional groups. Six carriers were verificated for DNA isolation: P (HEMA-co-GMA) ox, F-kol B 30 ox, F-kol 77 ox, F-kol B100 ox, F-kol 135 ox, coated with carboxyl groups and Perovskit 439 (coated by silicone). Bacterial DNA was isolated by phenol extraction procedure, first. DNA was reversibly bond to magnetis carrier in the presence of high concentration of NaCl ( 5 M) and poly (ethylene glycol) (PEG 6000). The final PEG and NaCl concentrations of 16.0 % (w/v) and 2.0 M, respectively, were used.DNA was eluted into TE buffer. The quality of extracted DNA was checked by PCR amplification. It was found out that although different quantities of DNA were isolated, the quality of isolated DNA was always compatible with PCR. Nanoparticles Perovskit 439 had the best separative characteristics in comparison to the other magnetic carriers because highest amounts of DNA was isolated. However, next optimisation of DNA separation procedure is required for the use of studied microspheres in real samples.
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