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. 2018 Dec 12;5(12):181369.
doi: 10.1098/rsos.181369. eCollection 2018 Dec.

Magnetofection and isolation of DNA using polyethyleneimine functionalized magnetic iron oxide nanoparticles

Adheesha N Danthanarayana  1 , Danushika C Manatunga  1 , Rohini M De Silva  1 , N Vishvanath Chandrasekharan  1 , K M Nalin De Silva  1   2
Affiliations

Magnetofection and isolation of DNA using polyethyleneimine functionalized magnetic iron oxide nanoparticles

Adheesha N Danthanarayana et al. R Soc Open Sci. .

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.

Keywords: DNA isolation; iron oxide nanoparticles; magnetofection; polyethyleneimine.

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Conflict of interest statement

We have no competing interests.

Figures

Figure 1.
Figure 1.
(a) FT-IR spectrum of polyethyleneimine functionalized iron oxide nanoparticles, (b) STEM image of PEI coated iron oxide nanoparticles and (c) TEM image of neat IONPs.
Figure 2.
Figure 2.
XRD pattern of PEI functionalized iron oxide nanoparticles.
Figure 3.
Figure 3.
Gel picture of plasmid DNA remaining in the supernatant when the added DNA content is in the range of 25–31 µg.
Figure 4.
Figure 4.
Gel picture of the remaining genomic DNA in the supernatant under optimized conditions.
Figure 5.
Figure 5.
Gel picture of plasmid DNA eluted with elution buffer 3 provided with heat at 60°C (1) control, (2) supernatant after binding DNA, (3) washing 1, (4) washing 2, (5) elution 1, (6) elution 2, (7) elution 3.
Figure 6.
Figure 6.
Gel picture of genomic DNA elution with elution buffer 4 heated up to 60°C (1) control, (2) supernatant after binding DNA, (3) washing 1, (4) washing 2, (5) elution 1, (6) elution 2, (7) elution 3.
Figure 7.
Figure 7.
Gel picture of plasmid DNA eluted from PEI-IONPs (1) control, (2) supernatant after binding DNA, (3) washing 1, (4) washing 2, (5) elution 1, (6) elution 2, (7) elution 3.
Figure 8.
Figure 8.
Gel picture of the PCR for isolated genomic DNA (1) marker (500 bp), (2) test sample 2, (3) test sample 1, (4) negative control.
Figure 9.
Figure 9.
(a) Optical microscopic images of (i) cells transfected with PEI and DNA, (ii) cells transfected with PEI/iron oxide nanoparticles and DNA, (iii) untransfected cells, (b) fluorescence microscopic images of (i) untransfected cells, (ii) cells transfected with PEI and DNA, (iii), (iv) cells transfected with PEI/iron oxide nanoparticles and DNA.
See this image and copyright information in PMC

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