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Genom1C Dna Extraction From Plant Tissue

Plant cells contain three types of DNA - nuclear, mitochondrial, and chloroplast DNA. Isolating total DNA from plant tissue requires removing cell walls and membranes, separating DNA from other components, and protecting it from degradation. The main challenges are DNAase enzyme activity that can degrade the DNA and other macromolecules that copurify with or bind to the DNA. EDTA is used to chelate cations needed for DNAase activity, and SDS detergent inhibits enzymes and dissociates proteins from DNA. For some plants, PVP is added to complex with polyphenols that can interfere. CTAB detergent isolates DNA by forming a soluble complex with DNA in high salt concentrations that precipitates DNA away from other compounds

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Sudheer Aluru
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39 views1 page

Genom1C Dna Extraction From Plant Tissue

Plant cells contain three types of DNA - nuclear, mitochondrial, and chloroplast DNA. Isolating total DNA from plant tissue requires removing cell walls and membranes, separating DNA from other components, and protecting it from degradation. The main challenges are DNAase enzyme activity that can degrade the DNA and other macromolecules that copurify with or bind to the DNA. EDTA is used to chelate cations needed for DNAase activity, and SDS detergent inhibits enzymes and dissociates proteins from DNA. For some plants, PVP is added to complex with polyphenols that can interfere. CTAB detergent isolates DNA by forming a soluble complex with DNA in high salt concentrations that precipitates DNA away from other compounds

Uploaded by

Sudheer Aluru
Copyright
© Attribution Non-Commercial (BY-NC)
We take content rights seriously. If you suspect this is your content, claim it here.
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GENOM1C DNA EXTRACTION FROM PLANT TISSUE

Plant contains three types of DNA: nuclear, mitochondrial and chloroplast DNA. Although quite elaborate exist for the isolation of each type of DNA, most experiments require only the rather simple preparation of total DNA. All DNA preparation methods involve the removal of cell wall and nuclear membranes, the separation of DNA from all other components, and the protection of the DNA during the procedure from nucleases and mechanical shearing. The problem in isolation DNA from plant, is the presence of DNAase activity which degrade the DNA and the presence of other macromolecules which co-purity with, or polymerize to the DNA during the isolation procedure. The nuclease problem is reduced by removing cations such as Mg+^ which are required for nuclease activity using EDTA. In addition detergent agent such as sodium dodecyle sulphates (SDS) are often used to inhibit enzyme activities, to dissolve membranes, and to dissociate proteins from DNA to make them more accessible to degradation.
Plant researches often encounter undesirable macromolecules, other than DNA, which create problems in the DNA isolation procedure. In some plant like beans, grapes, etc, it necessary to add 5% polyvinyl polypyrolidone (PVP) to the extraction buffer to purge polyphenols. PVPP forms complex hydrogen bonds with polyphenolic compounds that can be separated from DNA by centrifugation. PVPP is usually not necessary to isolate DNA from grasses. Isolation of DNA from plant is preferably achieved by treatment with CTAB . This detergent carries a positive charge that interacts with the negative charge of DNA in high salt concentration and form a soluble complex. In subsequent steps a decrease in the salt concentration causes precipitation of DNA, leaving other compounds, especially polysaccharides, in solution. The CTAB itself is removed by chloroform extraction leaving DNA in the aqueous phase to be ethanol precipitated.

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