An improved method of DNA isolation suitable for PCR-based detection of begomoviruses from jute and other mucilaginous plants

Isolation of High-Quality DNA from a Desert Plant Reaumuria soongorica

“…During the extraction, 3 mol/L sodium acetate (NaAc) added combined with chloroform/isoamyl alcohol extraction can reduce markedly the coprecipitation of polysaccharides with the nucleic acids and remove most proteins, polysaccharides, polyphenols and other impurities for the first time. The crude nucleic pellet was dissolved in 1 ml of 1 M NaCl instead of dissolving it in Tris-EDTA (TE), which ensured further reduction of viscosity of the mucilaginous substances (Chen and Chen, 2004;Ghosh et al, 2009). DNase-free RNase was added to crude DNA samples dissolved in1 ml of 1 M NaCl to completely clear residual RNA.…”

“…Now there are many extraction methods of genomic DNA from biomaterials. The commonly employed DNA isolation methods involve the use of enzymes such as lysozyme and proteinase K (Lockhart et al, 1989), cetytrime-thylammonium bromice (CTAB) (Ghosh et al, 2009;Moyo et al, 2008;Khanuja et al, 1999;Novaes et al, 2009;Singh et al, 2000) or sodium dodecyl sulfate (SDS) (Kaufman et al, 1999;Dellaporta et al, 1983) treatment and extraction with organic solvents, detergent-induced lysis in conjunction with proteinase K and lysozyme (Perera et al, 1994) or lysis using guanidinium isothiocyanate (GITC)-containing solutions (Boom et al, 1990;Noordhoek et al, 1995;Chakravorty & Tyagi, 2001), among which, guanidinium thiocyanate has been shown to be a powerful agent in the purification of DNA because of its potential to lyse cells and its potential to inactivate nuclease (Boom et al, 1990;Chomczynski et al, 1987;Zeillinger et al, 1993). However, high amounts of gummy polysaccharides, polyphenols and other various secondary metabolites such as alkaloids, flavonoids, terpenes and tannins in the desert plants usually hamper the DNA isolation procedures and reactions such as DNA restriction, amplification and cloning (Moyo et al, 2008;Khanuja et al, 1999;Pang et al, 2011;Zhang K., 2011;Ji & Li, 2011).…”

Detection of Corchorus golden mosaic virus Associated with Yellow Mosaic Disease of Jute (Corchorus capsularis)

“…Total DNA was isolated from 100 mg leaf tissues of experimentally inoculated jute plants maintained under glasshouse and field samples as per modified CTAB method [13]. Total DNA was also isolated from groups of 10 viruliferous whiteflies following CTAB method [9].…”

Molecular Characterization of a Jatropha leaf curl virus (JLCuV) Infecting a New Host Ludwigia parviflora in India

“…Total DNA was extracted from the symptomatic leaves using CTAB method (Ghosh et al, 2009), tested for the presence of geminiviruses by PCR using indigenously designed geminiviruses specific degenerate primer pair (Roy et al, 2015).…”