Abstract
For fast and easy isolation of inhibitor-free genomic DNA even from the toughest plant leaf samples, including those high in polyphenols and polysaccharides, a protocol has been developed. To prevent the solubility of polysaccharides in the DNA extract, high salt concentration (1.4 M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) was used for the removal of polyphenols as polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by proteinase K and removed by centrifugation from plant extracts during the isolation process resulting in pure DNA and RNA ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA and RNA isolated from leaves and roots of recalcitrant plants which was free from contamination and color. The average yields of total RNA from roots and shoot of Betula and Grape ranged from 285 to 364 ng/µl with A260/A280 between 1.9 and 2.08. The RNA isolated with this protocol was verified to be suitable for PCR, quantitative real-time PCR, semi-quantitative reverse transcription polymerase chain reaction, cDNA synthesis and expression analysis. This protocol shown here is reproducible and can be used for a broad spectrum of plant species which have polyphenols and polysaccharide compounds.
Highlights
Proteins like various enzymes were degraded by proteinase K and removed by centrifugation from plant extracts during the isolation process resulting in pure DNA and RNA ready to use in downstream applications including PCR, quantitative polymerase chain reaction, ligation, restriction and sequencing
The isolation of high-quality DNA and RNA is important in any molecular biology work because contaminants such as proteins, polyphenols and polysaccharides may interfere with enzymes such as restriction enzymes and Taq polymerase [in polymerase chain reaction (PCR)] (Angeles et al 2005)
DNA samples are often contaminated with polysaccharides, polyphenols, which are almost insolvable in water or Tris–EDTA (TE) buffer and are difficult to separate from DNA and RNA
Summary
The isolation of high-quality DNA and RNA is important in any molecular biology work because contaminants such as proteins, polyphenols and polysaccharides may interfere with enzymes such as restriction enzymes (in blotting techniques) and Taq polymerase [in polymerase chain reaction (PCR)] (Angeles et al 2005). DNA samples are often contaminated with polysaccharides, polyphenols, which are almost insolvable in water or Tris–EDTA (TE) buffer and are difficult to separate from DNA and RNA. These contaminants are readily identified as they impart a sticky gelatinous brown color to the DNA isolated and interfere with polymerases, ligases and restriction enzymes (Ogunkanmi et al 2008). Plant metabolites such as polysaccharides have a similar structure of nucleic acids and are not efficiently removed by most homebrew DNA and RNA isolation methods.
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