SALICYLIC ACID COATED IRON OXIDE MAGNETIC NANOPARTICLES FOR GENOMIC DNA EXTRACTION FROM COFFEE (Coffea arabica L.) LEAF

Abstract

Currently, DNA extraction using magnetic nanoparticles (MNPs) has gained significant consideration due to their simple manipulation, cost effectiveness and less time consumption. Although magnetic separation has been extensively studied for different biological samples, there is a current need for efficient methods to extract DNA from plants. The present study was aimed at synthesizing Fe3O4 MNPs coated with salicylic acid (SA) for gDNA extraction from coffee leaves. The Fe3O4 MNPs were synthesized from iron salts under alkaline conditions via the coprecipitation method, followed by coating using salicylic acid. The synthesized materials were characterized by ultraviolet-visible (UV-Vis) spectroscopy, x-ray diffraction (XRD) crystallography, Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). The point of zero charge was determined using the pH drift method. The Fe3O4, Fe3O4/SA MNPs and CTAB methods were used for gDNA extraction from coffee leaves. The experiment was done in a completely randomized design with three replications. The gDNA quality and quantity were examined using UV-Vis spectrophotometer, agarose gel electrophoresis, and PCR assays. The extraction efficiency of Fe3O4/SA MNPs was compared with that of Fe3O4 MNPs and the CTAB method. Moreover, the pH of the solution, the MNP dose, the incubation time, temperature and the pH of the elution buffer were optimized for the extraction. The DNA adsorption and desorption efficiency were also determined using the known volume and concentration of gDNA. The Fe3O4 MNPs had strong absorption peaks at 283 nm and 381 nm, while the Fe3O4/SA MNPs exhibited peaks at 235 nm and 295 nm. XRD analysis showed that the crystallite phase sizes of Fe3O4 and Fe3O4/SA MNPs were 14.35 and 10.44 n respectively. As per the FTIR finding, Fe-O (566 cm-1 ) was found in Fe3O4 and Fe3O4/SA MNPs, whereas C=O (2343 cm-1 ) and C-O (1126 cm-1 ) found in Fe3O4/SA MNPs only. The SEM analysis demonstrated that Fe3O4 and Fe3O4/SA MNPs appeared to have regular sizes of 20 µm and 5 µm, respectively. The Fe3O4 and Fe3O4/SA MNPs were found to have points of zero charges of 5.0 and 4.46 respectively. The extraction results revealed that the quality and quantity of extracted gDNA using Fe3O4/SA MNPs were higher than those of the bare Fe3O4 and CTAB methods. The PCR assay indicated the success of the extracted gDNA amplification that showed the absence of inhibitors of PCR. In addition, pH 4, 0.3 mg, pH 10, 65 °C, and 15 min were identified as the optimum pH of the solution, MNP dose, pH of the elution buffer, incubation temperature and time respectively. The highest adsorption (80%) and desorption (67%) efficiency were observed for Fe3O4/SA MNPs. The results revealed the effectiveness and convenience of the synthesized Fe3O4/SA MNPs in gDNA extraction from coffee leaf samples using simple and cost effective materials. Therefore, it can be recommended for gDNA extraction from plant samples even where sophisticated laboratory facilities are inaccessible.