Plant Biotechnology

LABORATORY EXERCISE
Isolation of Genomic DNA from Plant Tissues

Introduction
There are many difficulties associated with the isolation of undergraded plant nucleic
acids which are free contaminating proteins and polysaccharides. Different
extraction methods are necessary since different plant groups contain diverse
secondary compounds which may interfere with the isolation. Most plant cells have
very tough cell walls. Therefore vigorous methods are required to break cells open.
The efficiency of the physical force exerted on a tissue is critical for a good recovery,
but excessive force degrades very high molecular weight molecules through
shearing.

Plant tissues contain active nucleic acid degrading enzymes and the time between
cell rupture and inactivation of these nucleuses is a critical factor in the isolation of
intact DNA and RNA. Tissues with high carbohydrate content may benefit from
extraction with larger volumes of buffer. Polysaccharides may be removed by
binding to ethidium bromide (EtBr) or by precipitation with cetrimonium bromide
(CTAB).

Isolation of DNA can be described by two methods. In first method, plant cells are
lysed with ionic detergent, treated with protease, and subsequently purified by
cesium chloride (CcSl) density gradient centrifugation. The second method is a based
upon a series of treatments with the nonionic detergent cetrimonium bromide
(CTAB) to lyse cells and purify nucleic acid. Nucleic acid is recovered from the final
CTAB solution by isopropanol or ethanol precipitation. The first method, although
somewhat more lengthy, results in highly purified nucleic acids. The second method
requires fewer manipulations, results in very high yields, and produces DNA that is
less pure but nonetheless suitable in quality for use in many molecular biology
manipulations.

Materials
Young leaves of Canna glauca and Muricata paniculata; flower bud of Zinnia elegans;
mortar and pestle; liquid nitrogen; 2x CTAB buffer; 1.5 mL centrifuge tube;
chloroform-Isoamyl alcohol (CIA; 24:1); isopropanol; wash buffer; TE buffer.

Procedures
1. 0.1g of fresh young leaves of Canna glauca and Muricata paniculata, and flower
bud of Zinnia elegans; was weighed and was cut into smaller pieces.
2. The tissue was frozen rapidly in liquid nitrogen and using a mortar and pestle, the
tissue was grinded into powdery form.
3. 1 mL of pre-heated 2x CTAB buffer was added using titration method from
micropipette and the grinding process was continued until slurry was formed.
4. The mixture was transferred into a fresh 1.5 mL centrifuge tube.
5. The samples were incubated for 30-45 mins at 650C in a water bath.
6. 400 μl of CIA was then added. The content was mixed gently but thoroughly by
inverting the tube.

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7. The sample was then centrifuged at 13000 rpm for 5 minutes to obtain
supernatant.
8. The upper aqueous layer was transferred to a new 1.5 ml centrifuge tube.
9. Equal volume of ice-cold propan-2-ol (isopropanol) was added and mixed gently.
The tube was left to stand at -20°C for at least 30 mins.
10. The pellet of the DNA was obtained by centrifuging at 13000 rpm for 2 minutes.
11. The supernatant was carefully discarded and 1mL of wash buffer was added. The
sample was mixed gently for 5 mins.
12. The sample was centrifuged again at 13000 rpm for 2 minutes.
13. The supernatant was discarded carefully and the pellet was air-dried.
14. The pellet was dissolved in 50 µl of TE buffer and stored at -20°C until further
use.

Discussion
Use of juvenile sample
In the extraction of DNA samples, usually the use of young juvenile plant samples is
adopted. This serves as many benefits. This is mainly because they are young, softer,
less differentiated than old, and much matured samples. This makes the tissue
grinding process easier. Also involves in fewer steps of isolation. Younger samples
also have better quality of DNA recovery. Additional benefits are the reduced
chances of polysaccharide contamination that is usually the reason behind
unsuccessful DNA extraction of old samples. Contamination of polysaccharide
interferes by inhibiting the restriction enzymes and other DNA modifying enzymes.
Older plant samples also have more phenolic compounds that interfere with DNA
extraction.

Functions of the chemicals

Liquid nitrogen used to remove cell wall and produce fine grinds (the finer the grind,
the greater the yield) while keeping harmful chemicals and cellular enzymes
deactivated, thus reducing shear and damages to the DNA.

The CTAB (cetrimonium bromide) is a cationic surfactant and serves two purposes:
the detergent component in CTAB is used to disrupt the cell membrane and nuclear
membrane to expose the genetic components, once this happens the proteinase in
CTAB frees the DNA by destroying the histones that the DNA is wrapped tightly
around. This is allowed to take place at 65°C to optimize the enzyme activity in the
CTAB.

The use of CIA (chloroform-isoamyl alcohol) is to remove the proteins, most lipids,
and cellular debris that can cause an impure DNA result. It does so by binding with
non-aqueous compounds and separates it from the aqueous compounds.
Centrifuging this will create a precipitate of clumped proteins, non-aqueous lipids
and cellular debris, whereas the DNA is extracted from the supernate.

The isopropanol is added afterwards so that the DNA uncoils and comes together
since it is insoluble in alcohol. When it is initially added, DNA strands are visible. It

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produces a pellet after another centrifugation process. Isopropanol also removes

alcohol soluble salts.

The removal of any remaining salt or alcohol residues is achieved by washing the
pellet DNA with a wash buffer. This is to remove any remaining traces of impurities
and thus avoiding contamination.

TE buffer acts as a chelation cation for the DNA. It binds to the DNA and protects it
from further degradation and shearing, which stops the result of incorrect DNA
results or shortened DNA strand.

Tips
It is good to prepare all chemicals and materials needed as to avoid wasting time
and human error. Gloves and safety goggles should be worn to avoid chemical
irritation. Phenol is a dangerous chemical and should be used under the hood.

The use of liquid nitrogen is advisable as it deactivates the enzyme activity and thus
reduces chances of broken DNA strand. It is best to grind it as fine as possible as
this shows high yield of DNA.

Before centrifugation is best to invert the vial tube several times but not as
vigorous as shaking it. This is to allow even distribution of the added solution so
that it is well mixed and function maximized. After inverting the tubes several times
it is good to open the cap slowly and carefully as to remove pressure, this helps
uniform mixing. After centrifugation, be careful as to avoid remixing the aqueous
upper layer with the particulate layer below it.

The wash buffer can be used twice to increase assurance of purity.

When extracting the DNA supernate, do not immerse the micropipette too deep.
Avoid sucking up the non-aqueous particles, as going too deep with the
micropipette can cause in sucking up the protein and debris that is a source of DNA
contamination.

For high yield of DNA with minimal contamination, it is best to use young fresh
plant samples. But it the case that is not possible, storing tubes in a refrigerator-
freezer for 3-5 days in isopropanol increases DNA precipitation and thus increasing
the final DNA yield. Cold isopropanol is used to avoid degradation of DNA. A low
concentration can be used to further avoid the degradation but it would then be
best to leave this to sit over night for a more effective DNA precipitation.

To facilitate the action of the TE buffer, it can be soaked in hot-water bath for 5
minutes.

Conclusion
From this experiment, we become aware and understood on how isolation of
genomic DNA of plant tissues works.

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References
Croy, R.R.D. 1993. Plant Molecular Biology. BIOS Scientific Publishers, London, UK.

Doyle, J. J. and Dickson, E.E. 1987. Preservation of Plant Samples for DNA
Restriction Endonuclease Analysis. Taxon. 36: 715-722.

Doyle, J. J. 1991. DNA Protocols for Plants. In Hewitt, G., Johnson, A.W.B., and Young,
J.P. (eds), Molecular Techniques in Taxonomy. pp 283-293. NATO Scientific
Affairs Division, England, UK.

Henry, R.J. 2001. Plant Genotyping: The DNA Fingerprinting of Plants. CABI
Publishing, Oxon, UK.

Lin, J.Z. and Ritland, K. 2007. Flower Petals Allow Simpler and Better Isolation of DNA
for Plant RAPD Analyses. Plant Molecular Biology Reporter. 13 (3): 210-213.

Richards, E., Reichardt, M., and Rogers, S. 1994. Preparation of Genomic DNA in
Plant Tissue. In Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D.,
Seidman, J.G., Smith, J.A., and Struhl, K. (eds), Current Protocols in Molecular
Biology. pp 50-57. John Wiley & Sons, New York, USA.

Weising, K. 2005. DNA Fingerprinting in Plants: Principles, Methods, and Applications.

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