LANZUELA, Millicent Bernadette S.

C-2 BIO 101 - Z

DNA ISOLATION
 Process of extracting DNA from the cell through disruption of cell wall or cell membrane
and nuclear membrane through various methods such as chemical, enzymatic or
mechanical procedures.
 DNA can be extracted from number of sources such as human hair, saliva, urine, plant
tissue or cells as well as on animal tissues and cell.
Major procedures in DNA Isolation (PLANT TISSUES)
Sample Preparation and Lysis
 In plant samples, tissues are usually used to obtain the DNA. As plant cells have
rigid cell wall, plant samples are prepared by physical method such as grinding
the tissue using mortar and pestle with liquid nitrogen. The plant sample can be
easily turned into fine powder when frozen which is also the most suitable form
for extracting DNA.
o In the study of Nunes, et. al. (2011) strawberry leaves sample were
underwent tissue lyophilization and maceration with polyvinylpyrrolidone
(PVP).
 After the plant tissue has been powdered, it is then transferred to Eppendorf tube
and suspended to a CTAB lysis buffer, a buffer commonly used for plant sample.
 Vortex can be used to thoroughly homogenize the sample.
 Incubate the sample to a hot water bath for 30 minutes or an hour. Allow to cool
to room temperature.
DNA Precipitation
 After the cell has been disrupted and DNA is freed from the nuclear membrane,
other parts of the cell that have broken up are dispelled in the together with the
DNA.
 A volume ratio of (24:1) chloroform: isoamyl alcohol can be added to the
homogenate and centrifuge. This separates the DNA from the cell debris.
 Two distinct phases can be obtained wherein the upper aqueous layer that
contains the DNA will be used and transferred to a new tube.
 To further remove contamination, the extraction can be repeated until the aqueous
layer is clear.
 Gently homogenize the sample with an equal volume of cold isopropanol is added
and incubate at -20 ̊C.
DNA Purification
 Centrifuge the sample and carefully discard the supernatant such that the pellet
formed will not be agitated.
  Wash and centrifuge the pellet, twice, with 70% ice cold ethanol, and allow to air
dry. Washing removes the extra salt still suspended in the solution.
 Resuspend and dissolve the pellet, which is now the purified DNA, in TE buffer
or pure water for testing and keep in a hot air oven at 65 ̊C.
 Cool at room temperature and keep in refrigerator at 4 ̊C for storage.
 After the DNA is isolated, it can be tested to quantify and qualify its purity.
Major procedures in DNA Isolation (ANIMAL CELLS)
Preparation of cell extract
 The preparation for the animal tissue to be used do not largely differ from the
preparation done for plant tissue.
 For tissue samples, it can be cryogenically ground into fine powder using liquid
nitrogen or an ice bath. Digestion buffer is added and store at room temperature.
 For cell samples such as goat liver cells, a digestion buffer can be added directly
to the culture dish. When a pellet is formed, add another extraction buffer and lyse
the cells by repeated pipetting.
Purification of DNA
 Add 10% SDS and proteinase K in the homogenate and subject to vortex.
 Incubate the sample in 55 ̊C, then centrifuge and discard the supernatant formed.
 Put the microtube in an ice cold-water bath and consequently add chloroform and
6 M saturated NaCl in the microtube.
 After gently shaking the microtube for 30 sec, centrifuge until completely sorted.
 Transfer the supernatant to a new tube and add cold absolute ethanol and
ammonium acetate. Gently vortex the solution and DNA take its appearance as a
white spirals/coils. Centrifuge the homogenate again as 4 ̊C.
 Until a pellet is formed, discard the supernatant and let the pellet dry at room
temperature.
 For storage, the pellet can be dissolved in distilled water and incubate at -20 ̊C
until use.
Measurement of purity and DNA concentration
 DNA concentration and purity are measured by assessing aliquots on 0.8%
agarose gel and reading absorbance with a spectrophotometer at 260 nm.
REAGENTS/CHEMICALS USED
 CTAB isolation buffer – a cationic buffer typically used for plant cell disruption by
interfering with the membrane proteins.
o 2% CTAB or cetyl trimethyl ammonium bromide disrupts the membranes.
o 1.4 M NaCl - influences the solubility of DNA and other molecules present in the
extraction.
o 0.2% 2-mercaptoethanol – a reducing agent that aids in denaturing proteins
through cleavage of disulfide bonds between cysteine residues, removing tannins
and polyphenols present in the crude extract.
o EDTA – a chelating agent that leads to formation of chelate with magnesium ions
for DNase activity. Mg2+ acts as the cofactor of DNase, thus its absence result to
deactivation of DNase thus, prevents degradation of DNA.
o Tris HCl – neutralizing agent that aids in the precipitation that acts on the
negative charges in the DNA for molecules to come to bundle up.
 chloroform: isoamyl alcohol (24:1) – prevents frothing and esterification of lipid.
o Chloroform improves efficiency of nucleic acid extraction by denaturing protein,
aids in the dissociation of protein from nucleic acid apart from catalysis of
removal of lipid, while isoamyl acts an anti-foaming agent.
o When present in large concentrations, they cause dehydration of DNA by
extracting water.
 isopropanol and ethanol (85 and 75%) – aside from precipitating DNA, it creates a
hydrophobic environment as it displaces water of hydration required for solubilization of
DNA.
 Polyvinylpyrrolidone – protect against phenolics in which it binds to proteins and DNA
through hydrogen bonds immediately after plant cells are lysed.
 TE buffer (pH 8.0) - solubilizes DNA, while protecting it from degradation
o Tris – stabilizes and maintain pH at 8 during cell lysis, preventing DNA
degradation.
o EDTA – chelating agent that further inactivates nucleases, by binding to metal
cations.
 Digestion buffer – maintains the integrity of the DNA from lysis and maintains the pH
during extraction in cases such as acidic degradation.
o Components include Na2EDTA, Tris-HCL, NaCl.
 10% SDS – an anionic detergent commonly used in animal cell disruption which, like
CTAB buffer, hinders the membrane proteins and lipid layer which results to disruption
of the membranes such as the nuclear membrane.
 proteinase K – helps in the breakdown of peptide bonds.
 6 M saturated NaCl – diluting a nucleic acid together with an alcohol, it precipitates
nucleic acid spontaneously and can be pelleted by centrifugation.
 Distilled water – commonly used as solvent
REFERENCES
Das, J. Isolation of genomic DNA from goat liver cells. B.Sc. Part – III (Honours)
Henry, R. J. 2001. Plant Genotyping: the DNA Fingerprinting of Plants. Plant DNA extraction.
CAB International. DOI: 10.1079/9780851995151.0239.
Nunes, F. C., Ferreira, J. L., Fernandes, M. C. N., Breves, S. S., Generoso, A. L., Soares, B. D.
F., Dias, M. S. C., Pasqual, M. Borem, A., Cancado, G. M. A. An improved method for genomic
DNA extraction from strawberry leaves. Ciência Rural, Santa Maria. Vol. 41, n. 8: pp. 1383 –
1389.
Wang, T. 2011. A simplified universal genomic DNA extraction protocol suitable for PCR.
Genetics and Molecular Research. 10 (1): pp 519-525.
Yari, P. Valiee. T, Bashiri, H. Kahrizi, D. Yari, K. 2017. An Efficient and Optimized Protocol
for DNA Extraction from Animal Tissues Biological, Environmental and Agricultural Sciences
2: pp. 41-44.