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 Zinnia elegans ; Young leaves of Canna glauca and Muricata paniculata; flower bud of Zinnia mortar and pestle; liquid nitrogen; 2x CTAB buffer; 1.5 mL centrifuge tube; chloroform-Isoamyl 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 Zinnia elegans; was weighed and was cut into smaller pieces. bud of Zinnia 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 micropipette and the grinding process was continued until slurry was formed. 4. The mixture was transferred into a fresh 1.5 mL centrifuge tube. 0 5. The samples were incubated for 30-45 mins at 65 C 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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Plant Biotechnology
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. m ins. 12. The sample was centrifuged centrifuged again at 13000 rpm for f or 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 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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Plant Biotechnology
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. irritation. Phenol is a dangerous chemical and should be used under u nder 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. 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 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 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 refrigeratorfreezer 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. 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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Plant Biotechnology
References Croy, R.R.D. 1993. Plant Molecular Biology . BIOS Scientific Publishers, London, UK.
Doyle, J. J. and Dickson, E.E. 1987. Preservation Preservation of Plant Samples for DNA Restriction 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. Prin ciples, Methods, and Applications. Weising, K. 2005. DNA Fingerprinting in Plants: Principles, CRC Press, Boca Raton, Florida, USA.
http://www.celltechnology.com/products/ http://www.celltechnology.com/products/Wash%20Buffer.htm Wash%20Buffer.htm (220909) http://www.eeob.iastate.edu/facul http://www.eeob.iastate.edu/faculty/WendelJ/dnaextr ty/WendelJ/dnaextraction.htm action.htm (230909) http://wheat.pw.usda.gov/~lazo/method http://wheat.pw.usda.gov/~lazo/methods/lazo/dnaplmy.html s/lazo/dnaplmy.html (230909) http://www.cilr.uq.edu.au/UserImages/Fil http://www.cilr.uq.edu.au/UserImages/File/Plant%20Genomi e/Plant%20Genomic%20DNA%20Extracti c%20DNA%20Extracti on%20by%20CTAB%20_2__Fiona.pdf on%20by%20CTAB%20_2__Fiona.pdf (230909) http://www.geneaid.com/Plasmid_DNA http://www.geneaid.com/Plasmid_DNA_Extraction_Kit-Q&A5.htm _Extraction_Kit-Q&A5.htm (240909) http://serc.carleton.edu/micro http://serc.carleton.edu/microbelife/resear belife/research_methods/geno ch_methods/genomics/dnaext.htm mics/dnaext.htmll (240909)
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