US and Canadian vistors, can request a FREE SAMPLE of our CTAB based SYNERGY™ 2.0 Plant DNA Extraction Kit.
Isolating Deoxyribonucleic Acid (DNA) from plant tissues can be challenging as the biochemistry between divergent plant species can be extremely different. Unlike animal tissues where the same tissue type from different species usually has similar characteristics, plant tissue can have variable levels of metabolites and structural biomolecules. Polysaccharides and polyphenols are two classes of plant biomolecules that vary significantly between species and are problematic when isolating DNA. Contaminating polysaccharides and polyphenols can interfere with manipulations of DNA following isolation.
The following protocols for isolating clean plant DNA, both start with a traditional approach using a cetyltrimethylammonium bromide (CTAB) buffer. At that point they diverge, the first protocol makes use of phenol and chloroform, and the second protocol uses a reverse solid phase extraction (i.e., capturing contaminants on a solid phase). Methods using phenol and chloroform are preferred for the isolation of high molecular weight DNA, however both chemicals are considered hazardous. Phenol can cause very serious chemical burns while chloroform is a known carcinogen. The CTAB method using solid phase extraction, avoids phenol and chloroform and is best used for assays where smaller DNA fragment sizes are acceptable.
Skip ahead to the protocols:
As mentioned, polysaccharides and polyphenols are problematic when isolating DNA from plant tissues. CTAB buffers are effective at removing polysaccharides and polyphenols from plant DNA preparations. CTAB (also called hexadecyltrimethylammonium bromide) is a cationic detergent that facilitates the separation of polysaccharides during purification while additives, such as polyvinylpyrrolidone, aid in inactivating polyphenols. CTAB based extraction buffers are widely used when purifying DNA from plant tissues. The hazard with traditional CTAB protocols is the protein component of plant lysates is usually removed using phenol and chloroform. These two solvents are generally considered hazardous. The solid phase protocol listed below is an alternative. CTAB is more than a surfactant and its properties can be used in several ways to purify DNA. One option for purifying DNA using CTAB exploits the different solubilities of polysaccharides and DNA in CTAB depending upon the concentration of sodium chloride. At higher salt concentrations (1.4 M), polysaccharides are insoluble, while at lower concentrations (600 mM) DNA is insoluble. Consequently, adjusting salt concentration in lysates with CTAB, polysaccharides and DNA can be differentially precipitated. Most methods use CTAB to remove polysaccharides, followed by protein removal and DNA separation using precipitation or spin columns.
Plant cells contain phenolic compounds, such as catechol, that are catalyzed by polyphenol oxidase to o-quinones. The o-quinones in turn can alkylate and inactivate proteins. Polyphenol oxidases are found in plastids (i.e., chloroplasts) while catechol is found in vacuoles. When plant cells and tissues are disrupted, the enzyme and substrate mix and generate the reactive o-quinones (which is associated with browning of damaged leaves and fruit). Therefore, homogenizing plant tissue yields reactive molecules that can potentially interfere with subsequent manipulation of the DNA. To avoid the production of o-quinones, phenolic precursors are captured by polyvinylpyrrolidone (PVP) that is present in the homogenization buffer. PVP binds strongly with aromatic compounds, such as catechol and subsequent polyphenols, and prevents the formation of reactive o-quinones. CTAB-based protocols tend to work very well, but with one significant disadvantage phenol/chloroform extractions are routinely used to separate protein from the DNA. As chloroform is carcinogenic, many institutions frown upon its use. Furthermore, phenol can cause serious chemical burns. The traditional protocol will be covered, as well as an alternative protocol that uses solid phase extraction. The solid phase extraction is the basis of the Synergy™ Plant DNA Extraction Kit.
This method is best for isolating high molecular weight DNA. Caution is needed when working with liquid nitrogen, chloroform, and phenol. Consult your organization’s safety guidelines when working with these hazardous materials.
Plant samples can be prepared by cryogenically grinding tissue in a mortar and pestle after chilling in liquid nitrogen. Freeze dried plants can be ground at room temperature. In either case, a fine powder is best for extracting DNA.
Protocol for higher quality DNA, using silica spin columns to further purify the DNA.
This method eliminates the use of phenol and chloroform but requires the use of a bead beater. Bead beaters such as the Tissuelyser, GenoGrinder®, MiniG®, FastPrep, and small dental amalgamators will work. Bead beating shears DNA, thus DNA isolated is typically 2-7 Kb, sufficient for most Next-Generation Sequencing (NGS) and Polymerase Chain Reaction (PCR).