BIOLOGY INVESTIGATORY PROJECT

TOPIC: Isolation of DNA from animal cell

NAME: XYZ
CLASS: 12
ROLL NO:
DONE UNDER: Reetom Paul
 CONTENTS
1. Acknowledgment………………..2
2. Certificate………………………..3
3. Introduction……………………..4
4. Sources………………………….5
5. Mechanism………………………6
6. Procedure………………………..8
7. Role of buffer components……...12
8. Application of DNA isolation…...15
9. Conclusion………………………17
10. Bibliography……………………18

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 ACKNOWLEDGEMENT
I would like to express my special thanks to my
biology teacher Reetom Paul Sir for his able guidance
and support in completing my project. I would also like
to extend my gratitude to our Principal ma'am " Sujata
Chatterjee" and vice principal sir "Aniruddha
Bhattacharya" for providing me with all the facilities
that were required

DATE: 27th November,2022 NAME:

CLASS: XII
SEC: A

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 CERTIFICATE
This is to certify that XYZ, a student of Class-XII-A
has successfully completed this Investigatory project
report titled “ISOLATION OF DNA FROM ANIMAL
CELLS” under the guidance of Mr. Reetom Paul
(Biology Teacher) in the academic year 2022-2023.
This report is a bonafide work done by the student and
has been submitted to G.D. Goenka Public School,
Dakshineswar, in partial fulfilment of Biology practical
examination conducted by CBSE, New Delhi for the
award of All India Senior School Certificate
Examination (AISSCE) in Science.
Signature of examiner
…………………………
Signature of Biology Teacher
……………………………..

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 INTRODUCTION

DNA isolation/extraction is a process of purification of

DNA from a sample using a combination of physical
and chemical methods. It is frequently a preliminary
step in many diagnostic procedures used to identify
environmental viruses and bacteria and diagnose
illnesses and hereditary diseases. The first isolation of
DNA was done in 1869 by Friedrich Miescher.
Methods used to isolate DNA depend on the source,
age, and size of the sample tissue. In general, they aim
to separate DNA present in the nucleus of the cell from
other cellular components. Isolation of DNA is needed
for genetic analysis, which is used for scientific,
medical, or forensic purposes.

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 SOURCES
Sources of DNA for the purpose of isolation are diverse
and plenty. Common sources for DNA isolation include
whole blood, hair, sperm, bones, nails, tissues, blood
stains, saliva, buccal (cheek) swabs, epithelial cells,
urine. Stored samples can come from archived tissue
samples, frozen blood or tissue, exhumed bones or
tissues.

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 MECHANISM
Genomic (chromosomal) DNA from bacteria, plant or
animal cell can be isolated by lysis method. For plant
cell or bacterial cell, first cell wall is mechanically
disrupted. The cell wall is broken enzymatically using
enzymes like lysozyme. After this, the cell membrane is
disrupted using SDS (Sodium Dodecyl Sulphate),
detergents and surfactants which dismantle lipid
structures. The next step is precipitation of DNA. This
step is done in order to separate DNA from the other
cellular debris. In this step, sodium acetate is used to
neutralize the negative charges on the DNA molecules.
This makes the DNA more stable and less water
soluble. After this, ice-cold isopropanol is added to
precipitate DNA from the aqueous solution. The next
step is purification step. Once the genomic DNA is
released in the solution, phenol-chloroform solution is
used to purify the sample. Phenol denatures the protein.
After centrifugation, denatured proteins stay in the
organic phase and DNA stays in the aqueous phase. The
chloroform removes phenol residues from solution. The
alcohol is added to remove any unwanted material and
cellular debris. The purified DNA is then dissolved
in the TE buffer.

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Image showing the steps of DNA isolation

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 PROCEDURE
MATERIALS REQUIRED: Bovine blood samples,
50ml polypropylene conical centrifuge tube, RBC lysis
buffer, ice, 250 µl of 10% SDS, 25 µl of proteinase K,
Tris saturated phenol, phenol, chloroform,
isoamylalcohol, 0.3 M Na-acetate, 70% ethanol.
1. PREPARATION OF CELL EXTRACT:
a) Blood samples are transferred into 50ml
polypropylene conical centrifuge tube. The volume of
each tube is made up to 50 ml with RBC lysis buffer
and kept in ice for 10-15 minutes.
b) Tubes are centrifuged at 3000 rpm for 10 minutes at
4°C and supernatant is decanted carefully. To this cell
mass 20ml of chilled 1x lysis buffer is added and the
tubes are incubated at 0°C ice water bath for 5 to 8 min
with intermittent mixing and centrifuged at 3000rpm for
10 min at 4°C.
c) The supernatant is discarded and the lysis of RBC is
repeated until clear white pellet appears.
d) The white pellet containing WBC is dissolved
thoroughly in 5 ml of DNA extraction buffer.
e) To this 250 µl of 10% SDS is added slowly.

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f) 25 µl of proteinase K (20mg/ml) is added to each
tube cell lysates are incubated overnight at 56°C for
proper proteinase K digestion.
g) Cell lysates are extracted with equal volume of Tris
saturated phenol (pH;7.8) by gentle mixing and
centrifuged at 10,000 rpm for 10 min at room
temperature.
h) The upper aqueous phase is extracted with phenol:
chloroform: isoamylalcohol (25:24:1) and once with
chloroform: isoamylalcohol (24:1).
2. PURIFICATION OF DNA FROM CELL
EXTRACT:
a) The DNA is precipitated from the aqueous phase by
double volume of isopropanol in presence of 0.3 M Na-
acetate (pH 5.2).

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3. CONCENTRATION OF DNA SAMPLE:
a) Precipitated DNA is spooled out in 1.5 ml
microcentrifuge tube, and precipitated with ice-cold
70% ethanol.
b) Pellet is air dried and finally dissolved in 200-300 µl
of 1x TE Buffer (10:1, pH 8.0) depending on the size of
the pellet.

4. MEASUREMENT OF INTEGRITY OF DNA

CONCENTRATION:
a) Integrity of the total DNA is checked by performing
agarose gel electrophoresis in 0.8% agarose gel and
visualizing through Gel documentation system.

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 ROLE OF BUFFER COMPONENTS
SDS
It is an anionic detergent that helps in the denaturation
of cell membrane protein.
PHENOL
DNA is insoluble in phenol because phenol is a less-
polar solution. On the other side, protein has both polar
and non-polar groups because of the long chain of
different amino acids. The amino acid side chains have
varied groups. Also, the folding of the protein into the
secondary, tertiary and quaternary structure relies on the
polarity of the amino acids. When we add phenol,
bonds between amino acids break leading to protein
denaturation.
CHLOROFORM
Chloroform increases the efficiency of phenol to
denature the protein. Here, chloroform allows proper
separation of the organic phase and aqueous phase and
keeps DNA protected into the aqueous phase.
Chloroform denatures the lipid as well.
ISOAMYL ALCHOHOL
In the phenol-chloroform DNA extraction method,
Isoamyl alcohol helps in reducing foaming between
interphase. It prevents the emulsification of a
solution. The liquid phase contains DNA and the
organic phase contains lipid, proteins and other
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impurities. The precipitated protein denatured and
coagulated between both these phases. This will create
the cloudy, whitish- foam between interphase. The anti-
foaming agent, isoamyl alcohol stabilized the interphase
by removing the foaming and increasing the purity of
DNA. Noteworthy, the isoamyl alcohol is also practiced
as a DNA precipitation agent in the later stage of the
extraction process.
TRIS SATURATED PHENOL
It maintains the pH of the solution and also
permeabilizes the cell membrane.
Na2EDTA
It is a chelating agent and blocks the activity of the
DNase enzyme.
CH3COONa (3M)
Sodium acetate salt is used in DNA extraction to
precipitate the DNA. In the solution, the positive charge
Na + of the NaAc competes with the positive charge of
water to bind with the negative charge of DNA. It
electrostatically interacts with the PO3– of the DNA and
decreases the hydrophilicity of the DNA. Notedly,
ethanol helps in this process by protecting the Na + and
PO3– complex from the water, making it less stable and
precipitating DNA.
MgCl2
Protects DNA from mixing with other cell organelles
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 APPLICATIONS OF DNA ISOLATION
The ability to extract DNA is of great importance to
studying the genetic causes of disease and for the
development of diagnostics and drugs. It is also
essential for carrying out forensic science, sequencing
genomes, detecting bacteria and viruses in the
environment and for determining paternity.
Some common applications include:
1. Functional Cloning: It is a molecular cloning
technique that relies on prior knowledge of the encoded
protein's sequence or function for gene identification. In
this assay, a genomic or cDNA library is screened to
identify the genetic sequence of a protein of interest.
2. Cloning of superior breeds: DNA extraction is used
to clone organisms with desirable traits.
3. DNA fingerprinting: DNA fingerprinting is a
technique for identifying and analysing differences in
DNA between individuals. It is used to identify
prospective criminal suspects, prove paternity and
establish familial ties, identify and protect the
commercial crop and livestock types as well as figure
out an organism's evolutionary history and the
relationships between different groupings of species.
4. Disease diagnosis: Analysing isolated DNA helps us
identify diseases/disorders.

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5. Genetic diversity studies: Genetic diversity is the
base for survival of plants in nature and for crop
improvement. It is important because it gives species a
better chance of survival.

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 CONCLUSION
Since the first DNA isolation was successfully done by
Friedrich Miescher in 1869 and the initial DNA
extraction developed from density gradient
centrifugation strategies by Meselson and Stahl in 1958,
many techniques for biomolecules purification has been
developed. DNA extraction has helped researchers and
scientists in manipulating subsequent molecular biology
analysis in order to have a better understanding in the
biological materials of the earth. Automating nucleic
acid extraction process is potentially beneficial for a
number of reasons including to reduce working time,
decrease labor costs, increase worker safety and at the
same time provides opportunity in increasing
reproducibility and quality of results. However,
improvement of the weaknesses for some of the
instruments needs to be conducted all the time. In the
mean time, an all-in-one biomolecules extraction
system, or the invention of a miniature and portable
extraction system can become a prospective
development in the future.

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 BIBLIOGRAPHY
1. https://www.sigmaaldrich.com/IN/en/technical-
documents/protocol/genomics/dna-and-rna-
purification/extraction-protocol-animal-tissue
2.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC278953
0/
3.
https://whatisbiotechnology.org/index.php/science/sum
mary/extraction/dna-extraction-isolates-dna-from-
biological-material
4. https://link.springer.com/chapter/10.1007/978-94-
009-0019-6_1
5. Green, Michael R., and Joseph Sambrook.
"Molecular cloning." A Laboratory Manual
4th (2012).Green, Michael R., and Joseph Sambrook.
"Molecular cloning." A Laboratory Manual 4th (2012).
6. Sambrook, Joseph, Edward F. Fritsch, and Tom
Maniatis. Molecular cloning: a laboratory manual. No.
Ed. 2. Cold spring harbor laboratory press, 1989.

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