Practical No 11 : Isolation of DNA extraction from plant tissue

Introduction:

Cetyltrimethylammonium bromide is a cationic detergent which solubilizes membranes and from

complex with DNA. After cell disruption and incubation with hot CTAB isolation buffer, proteins are
extracted with chloroform –isoamyl alcohol, and the CTAB-DNA complex is precipitated with
isopropanol. The pellet is then washed with ethanol, dried redissolved in a suitable buffer.

Materials:

DNA extraction buffer:100mM Tris.HCl, pH 8.0, 20 mM EDTA, 1.4 M NaCl, 4% Cetyltrimehylammonium

bromide (CTAB)

Β-Mercaptoethanol

Chloroform-Isoamyl alchohol (24:1)

Ice-cold Isopropanol

Washing solution: 70% ethanol, 10mM ammonium acetate

TE buffer: 10mM Tris.HCl, pH 8.0, 1mM EDTA

Liquid N2, mortar and pestle, water bath, centrifuge

Method:

15ml of extraction buffer was added in to a 50ml capped polypropylene tube .then the tube was kept
in a 60°C water bath for about 10-15 min for pre-warming.0.15g of young leaves were placed in the
motor and freezed at -80°C.The tissues were quickly ground with pestle until fine powder is
obtained.15μl β-mercaptoethanol (0.1% in the buffer) was added to the pre warmed extraction buffer
just before adding the grounded tissue. The powder was then transferred in to pre warmed isolation
buffer solution; the tube was caped and mixed gently. The tube was incubated for 30 min at 60°C in
the water bath with shaking. The tube was inverted every 10 min for gentle mixing (mixing should be
done gently to prevent breakage of DNA). One volume (15ml) of chloroform-isoamyl alcohol (24:1) was
added to the tube, caped and mixed (by hand or in rotary) for 10 min (mixing was done gently but
thoroughly to ensure mixing of two phases). The tube was centrifuge for 10 min at 5000g at room
temperature. The upper aqueous was transferred into a 50ml tube. The volume was measured
(depending on the desired purity of DNA required, the aqueous phase may be reextracted several
times with volume of fresh chloroform-isoamyl alcohol). 0.6 volume of ice-cold isopropanol was added
in to the tube. The tube was closed and mixing was done gently but thoroughly by inverting the tube
several times.( At this stage , the DNA-CTAB complex was precipitated as a whitish network. then DNA
were spooled out with a clean glass rod and transferred into a micro centrifuge tube). The DNA-CTAB
complex forming glass was kept in -40°C for 20-30 minutes. The tube was centrifuge at 5000g for 10
min at -4°C. The supernatant was discarded and 1.5ml of washing solution was again added. The pellet
was gently agaitated for a few minutes. . The tube was centrifuge at 5000g for 10 min at -4°C. The
supernatant was carefully drained without spilling the pellet and the tube was kept inverted on a tissue
paper for about 1 hour to dry. (Pellet should neither contain residual ethanol nor should they be too
dry, as it will affect the solubility of the pellet in TE). An appropriate volume of TE buffer added (100-
500 µl). The pellet was left to dissolve without agitation (Higher molecular weight DNA may take
several hours to dissolve). DNA were quantified and analyzed by agarose gel electrophoresis.

Observations

Absorbance values of the mixture (250 μl) of Extracted DNA (7.5 μl) + Distilled water (742.5 μl)

At, 260nm = 0.4395 and 275nm = 0.3808

Calculations & Results:

Quantification of the DNA sample

Amount of double strand DNA = (A260 ×Dilution factor ×50)/1000

= (0.4395 ×100 ×50)/1000

= 2.198 μg/μl

Calculation to check the purity of the sample

(A260 )/A275 = (0.4395 )/0.3808 = 1.154

Conclusion
The yield of DNA of leaf tissue extracted was measured using a UV-VIS spectrophotometer at 260 nm.
The purity of DNA was determined by calculating the ratio of absorbance at 260 nm to that of 275 nm

Amount of double strand DNA in the sample = 2.198 μg/μl

Considering the A260/A275 which is less than 1.5 it can be concluded that the extracted sample is not
pure.

Discussion

Isolation of plant nucleic acids for use for Southen blot analysis, polymerase chain reaction (PCR)
amplifications, restriction fragment length polymorphisms (RFLPs), arbitrary primed DNA
amplifications (RAPD, APPCR,DAF), and genomic library construction is one of the most important and
time-consuming steps. The degree of purity and quantity varies between applications. A good
extraction procedure for the isolation of DNA should yield adequate and intact DNA of reasonable
purity. The procedure should also be quick, simple and cheap and, if possible, avoid the use of
dangerous chemicals.

The extraction process involves, first of all, breaking or digesting away cell walls in order to release the
cellular constituents. This is followed by disruption of the cell membranes to release the DNA into the
extraction buffer. This is normally achieved by using detergents such as sodium dodecyl sulphate (SDS)
or cetyl-methylammonium bromide (CTAB). The released DNA should be protected from endogenous
nuclease. EDTA is often included in the extraction buffer to chelate magnesium ions, a necessary co-
factor for nucleases, for this purpose. The initial DNA extracts often contain a large amount of RNA,
proteins,polysaccharides, tannins and pigments which may interfere with the extracted DNA and
difficult to separate. Most proteins are removed by denaturation and precipitation from the extract
using chloroform and/or phenol. RNAs on the other hand are normally removed by treatment of the
extract with heat treated RNase A.

Mechanical lysis for DNA extraction

Lysis can be done either by mechanically or chemically. In this practical we used mechanical lysis
process. Mechanical lysis means disruption of cells using sonication, a pressure cell, homogenizer, or
bead beater. Mechanical lysis methods are economical and preferable for large-scale preparations
because the addition of chemicals is not required. However, mechanical lysis produces heat, which
needs to be controlled. Care should also be taken to avoid foaming, to prevent surface denaturation
and oxidation. In our practical Cell lysis was performed by hand, using mortar and pestle.

While the grinding the lysosomes in the cells will also be broken and the digestive enzymes including
DNases in them will be released to the solution, which may degrade the DNAs prior to the extraction.
To avoid this the sample was frozen or added with liquid nitrogen, which provides a low temperature
environment, where the DNases are inactive.

As important as DNA molecules are to life, they are still extremely fragile, and break apart easily when
removed from cells. To slow down the rate at which the DNA breaks up, we cool down the buffer
solution to near freezing. Chemical reactions always take place slower in cold solutions than in warm
ones, because there is a lot less energy around to make the reaction take place.

CTAB (Cetyl trimethylammonium bromide)

Polysaccharide-like contaminants are more difficult to remove. They can inhibit the
activity of certain DNA-modifying enzymes and may also interfere in the quantification of nucleic acids
by spectrophotometric methods .NaCl at concentrations of more than 0.5 M, together with CTAB is
known to remove polysaccharides. CTAB will combine with the polysaccharides and proteins and
chemically alter them. CTAB binds to the cell wall debris, proteins and polysaccharides in the
homogenate: once perform a phenol-chloroform extraction after the CTAB step, the
CTAB/debris/polysaccharide/protein mix is removed.

In Plant DNA isolation due to its high polyphenolic content, this may interfere with the DNA purity
especially for subsequent manipulations. Antioxidants are commonly used to deal with problems
related to phenolics such as 2 -mercaptoethanol, ascorbic acid, Bovine Serum Albumin, sodium azide
and PVP amongst others. Phenol extractions when coupled with SDS are also helpful.

Buffer Solution
TE buffer is a commonly used buffer solution in molecular biology, especially in procedures involving
DNA or RNA. "TE" is derived from its components: Tris, a common pH buffer, and EDTA, a molecule
chelating cations like Mg2+. The purpose of TE buffer is to protect DNA or RNA from degradation.

EDTA
Ethylenediaminetetraacetic acid, widely abbreviated as EDTA is a polyamino carboxylic acid and a
colourless, water-soluble solid. Its conjugate base is named ethylenediaminetetraacetate. It is widely
used to dissolve limescale. Its usefulness arises because of its role as a hexadentate ("si ligand and
chelating agent, i.e. its ability to "sequester" metal ions such as Ca2+ and Fe3+. After being bound by
EDTA, metal ions remain in solution but exhibit diminished reactivity.

Metal-EDTA complex

TrisHCl/EDTA buffer has 2 main functions, which includes the pH stabilization done by TrisHCl and
EDTA is a chelating agent and has great affinity with matel ions and Mg-ion present in DNase as a
cofactor and responsible for DNase action that degrade the DNA,here EDTA bind with Mg-ion and
nullyfy the action of DNase.

Β-Mercaptoethanol
2-Mercaptoethanol is often included in extraction buffers designed for plant DNA extraction, because
it is a strong reducing agent which can remove tannins and other polyphenols often present in the
crude plant extract. It may also help to denature proteins by breaking disulphide bonds between
cysteine residues.
Phenol/Chloroform
The cell wall, some carbohydrates, lipids and proteins associated with the disrupted cell wall dissolve in
the phenol/chloroform. DNA is hydrophilic so remains in the aqueous portion.

Reference

1. Doyle JJ and Doyle JL (1990). Isolation of plant DNA from fresh tissue. Focus 12: 13-15.
2. Jobes DV, Hurley DL and Thien LB (1995). Plant DNA isolation: a method to efficiently
removepolyphenolics, polysaccharides, and RNA. Taxon 44: 349-386.
3. Sharma KK, Lavanya M and Anjaiah V (2000). A method for isolation and purification of peanut
genomicDNA suitable for analytical applications. Plant Mol. Biol. Rep. 18: 393a-393h.

Practical No 11