Plant Genomic DNA Extraction by using CTAB method

Principle:
DNA extraction from plant tissue can vary depending on the material used. Essentiallyany
mechanical means of breaking down the cell wall and membranes to allow access tonuclear
material, without its degradation is required. For this, usually an initial grindingstage
withliquid nitrogen is employed to break down cell wall material and allow accessto DNA
while harmful cellular enzymes and chemicals remain inactivated. Once thetissue has
beensufficiently ground, it can then be resuspended in a suitable buffer, suchas CTAB. In
order to purify DNA, insoluble particulates are removed throughcentrifugation while soluble
proteins and other material are separated through mixingwith chloroform and centrifugation.
DNA must then be precipitated from the aqueousphase and washed thoroughly to remove
contaminating salts. The purified DNA is thenresuspended and stored in TE buffer or sterile
distilled water. This method has beenshown to give intact genomic DNA from plant tissue.
To check the quality of theextracted DNA, a sample is run on an agarose gel, stained with
ethidium bromide, andvisualised under UV light.

Role of CTAB in DNA extraction:

Isolating 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.

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.

Role of PVP in DNA extraction:

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.

Role of Chloroform in DNA extraction:

Chloroform (CHCl3) or trichloromethane is a nonpolar (hydrophobic) solvent, in which

nonpolar proteins and lipids get dissolved to promote the partitioning of lipids and cellular
debris into the organic phase, leaving isolated DNA protected in the aqueous phase.
Chloroform ensures phase separation of the two liquids because it has a higher density (1.47
g/cm3) and forces a sharper separation of the organic and aqueous phases, thereby assisting in
the removal of the aqueous phase with minimal cross contamination from the organic phase.

Role of Isoamyl alcohol in DNA extraction:

Chloroform comes in contact with the air and forms gas phosgene (COCl2, carbonyl
chloride), which is harmful. If we simply use chloroform only, the gas entrapment causes
foaming or frothing, it foams up between interphase during extraction process and makes it
difficult to properly purify the DNA, which is prevented when chloroform is used along with
isoamyl alcohol or isopentanol [(CH3)2CHCH2CH2OH] or octanol [CH3(CH2)7OH] by
preventing the emulsification of a solution. Isoamyl alcohol or isopentanol is not miscible in
the aqueous solution because it is a long-chain aliphatic compound, containing five carbon
atoms and stabilizes the interphase between organic and aqueous layer. The aqueous phase
contains DNA and the organic phase contains lipid, proteins, and other impurities.

Role of ammonium acetate and absolute ethanol in DNA precipitation:

The role of the salt in the extraction protocol is to neutralize the charges on the sugar
phosphate backbone of the DNA. Ammonium acetate is commonly used for precipitation of
nucleic acid along with ethanol. In solution, ammonium acetate dissociates into NH4+ and
[CH3COO]-. The positively charged ammonium ions neutralize the negative charge on the
phosphate groups on the sugar phosphate backbone of nucleic acids reducing repulsion
between DNA molecules, making the DNA molecule far less hydrophilic, and therefore much
less soluble in water. Water has a high dielectric constant, which makes it fairly difficult for
the NH4+ and PO4-3 ions to come together. This is affected by absolute ethanol, which has a
much lower dielectric constant, making it much easier for NH4+ and PO4-3 to interact with
each other, and make the nucleic acid less hydrophilic, causing the DNA to drop out of
solution

Role of 70% ethanol wash:

DNA precipitate is washed again with 70% ethanol to rinse excess salt that might come along
with the extraction buffers from the pellet, centrifuged, and ethanol is discarded, leaving
DNA in the precipitate. Precipitate is air-dried or vacuum-dried. Over drying should be
avoided as DNA converts B form to D form, which is difficult to dissolve later.
Role of Tris-EDTA buffer for storage of DNA:

TE buffer contains Tris (10 mM) and EDTA (1 mM), where Tris is the buffering component
and EDTA the chelating component. For DNA isolation, the pH is usually set to 7.5–8.5, the
slight alkalinity of TE buffer also prevents chances of acid hydrolysis that may further disrupt
the stability of DNA stored in water. The purpose of EDTA is to chelate Mg+2 ions in
solution necessary for DNase activity, thus protecting the DNA.

Materials
Plant tissue (1 gm)
CTAB buffer
Mortar and Pestle
Liquid Nitrogen
Microfuge
Absolute Ethanol (ice-cold)
70 % Ethanol (ice cold)
7.5 M Ammonium Acetate
55◦C water bath
Chloroform:Iso Amyl Alcohol (24:1)
Water (sterile)
Agarose
6x Loading Buffer
1X TAE running buffer
Ethidium Bromide solution

CTAB buffer 100ml

2.0 g CTAB (Hexadecyl trimethyl-ammonium bromide)
10.0 ml 1 M Tris pH 8.0
4.0 ml 0.5 M EDTA pH 8.0 (EthylenediaminetetraAcetic acid Di-sodium salt )
28.0 ml 5 M NaCl
1g Polyvinylpyrrolidone
40.0 ml H2O
Adjust all to pH 5.0 with HCl and make up to 100 ml with H 2O.

Procedure
- Grind 1 gm of plant tissue to a fine paste in approximately 2.5 ml of CTAB buffer.
- Transfer CTAB/plant extract mixture to a falcon tube.
- Incubate the CTAB/plant extract mixture for about 15-20 min at 55◦C in water bath.
- After incubation, spin the CTAB/plant extract mixture at 12000 rpm for 5 min to spin down
cell debris. Transfer the supernatant to clean microfuge tubes.
- To each tube add 250 μl of Chloroform:Iso Amyl Alcohol (24:1) and mix the solution by
inversion. After mixing, spin the tubes at 13000 rpm for 5 min.
- Transfer the upper aqueous phase only (contains the DNA) to a clean microfuge tube.
- To each tube add 50 μl of 7.5 M Ammonium Acetate followed by 500 μl of ice-cold
absolute ethanol.
- Invert the tubes slowly several times and incubate at -20◦C overnight to precipitate the DNA.
- Spin the tubes at 12000 rpm for 10 min and decant the supernatant carefully.
- Add 1 ml of 70% ethanol to each tube and mix well followed by centrifugation at 12000
rpm for 10 min. The supernatant was discarded and the step was repeated again.
- The DNA pellet was air-dried and dissolved in 50-60 μl of TE buffer.
- The concentration and purity of the DNA was checked spectrophotometrically by recording
the absorbance at 260 nm. The quality of the extracted DNA was checked by loading 5-10 μl
of the sample in 1% agarose gel electrophoresis.