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Abstract
This study was carried out to develop a simple and efficient method to isolate DNA directly from biological samples using iron oxide nanoparticles (IONPs) functionalized with polyethyleneimine (PEI). IONPs were synthesized via co-precipitation method followed with direct attachment of branched PEI. Nanoparticles were characterized using STEM, FT-IR spectroscopy and XRD analysis. The binding capacity of synthesized PEI-IONPs for plasmid and genomic DNA was assessed using purified DNA samples. In order to elute bound DNA, elution conditions were optimized, changing pH, salt concentration and temperature. Synthesized PEI-IONPs were subjected to isolation of DNA from bacterial cell culture and from human blood. PCR and magnetofection of the enhanced green fluorescence protein (EGFP) were carried out to verify the downstream applications of isolated DNA. The results indicated that the synthesized nanoparticles were of 5–10 nm. The binding capacity of PEI-IONPs for plasmid DNA and genomic DNA were 5.4 and 8.4 µg mg−1, respectively, which were even higher than the commercially available kits such as Mag-bind, MagJET and Magmax. The optimized condition for plasmid DNA elution was 0.1 M Tris HCl (pH 10.0), 1.5 M NaCl and 5% formamide, maintained at the temperature of 60°C. The optimized condition for genomic DNA elution was 0.1 M Tris HCl (pH 10.0), 1.5 M NaCl and 10% formamide, maintained at 60°C. PCR and magnetofection processes were successful. This study revealed that the magnetic separation of DNA using PEI-IONPs is a simple and efficient method for direct isolation of DNA from biological samples which can be then used in various downstream applications.
Highlights
Efficient methods of DNA isolation is an ever growing subject in much research, as purified DNA is extremely important in medical and biotechnological fields
(PBS, pH 7.5), Tris EDTA (TE buffer, pH 7.5) which was used as the binding buffer and washing buffer, 3 0.5x Tris borate EDTA (TBE), gel loading buffer, diluted ethidium bromide solution, ET buffer, lysis buffer (10 N NaOH, 20% SDS, autoclaved water), KAc solution, RBC lysis buffer, LB 1 buffer, LB 2 buffer, DB buffer and pcDNA 3.1 (5.4 kb) having the enhanced green fluorescence protein (EGFP) gene (Invitrogen) which was cloned in XL1-Blue E. coli were supplied by the Molecular Biology Laboratory in University of Colombo
FT-IR spectroscopy was carried out to confirm the formation of Cetyltrimethyl ammonium bromide (CTAB) coated iron oxide nanoparticles and the spectrum is given in electronic supplementary material, figure S1
Summary
Efficient methods of DNA isolation is an ever growing subject in much research, as purified DNA is extremely important in medical and biotechnological fields. The development of magnetic purification methods has attracted much attention, and in this context iron oxide nanoparticles are the most extensively studied due to their unique properties such as lower toxicity, biocompatibility and easy degradability. Iron oxide nanoparticles have permitted most suitable and safest nanomaterial for many in vivo applications including magnetic resonance imaging (MRI) [4,5,6], drug and gene delivery [5,7,8,9], bioseparation [10] and hyperthermia [5,11]. The bio-distribution of nanoparticles can be altered by applying an external magnetic field, and they are widely used for biomedical applications specially due to their dispersity without forming magnetic aggregates
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