DNA ISOLATION of

plant and animal

tissue

Presented by :- Shubhangi Dalai

Research Scholar
at ICFRE-TFRI, Jabalp
 CONTENT

S.no. Chapter

1. Introduction

Protocol for Genomic DNA Isolation

2.
for plants

Protocol for Genomic DNA Isolation

3.
from Animal Tissue or Cultured Cells
 What is DNA?

Discovered in 1869.
Structure was determined by Dr James Watson and Francis Crick in 1953.
Watson and Crick were helped by research done by Rosalind Franklin (who took an X-
ray photo of DNA’s structure) and Maurice Wilkins
DNA EXTRACTION
 DNA extraction, is the process of separating DNA from other cellular
components to obtain pure DNA for further analysis or experimentation. It
involves several steps, including cell lysis, DNA precipitation, and purification.
The specific methods and reagents used can vary depending on the sample type
and intended use of the purified DNA.
 The first isolation of DNA was done in 1869 by Friedrich Miescher.
 Purpose of DNA Extraction :
To obtain DNA in a relatively purified form which can be used for further
investigations, i.e. PCR, sequencing, etc.
Why is DNA Isolation Important?

DNA isolation is a crucial step in many molecular biology

and genetic analysis techniques, including:
 DNA sequencing: To determine the sequence of DNA
bases.
 PCR (Polymerase Chain Reaction): To amplify
specific DNA sequences.
 Genetic testing: To identify genetic mutations or
variations.
 Forensic science: To analyze DNA samples from crime
scenes.
 Research: To study genes and their functions.
DNA Isolation methods :

 Phenol-chloroform extraction: a traditional method involving organic solvents to

separate DNA from proteins and other contaminants.
 Bead-based extraction: uses magnetic or other beads to bind to the DNA,
allowing for efficient separation and purification.
 Salting out procedures: utilizes high salt concentrations to precipitate proteins,
leaving the DNA in solution.
 CTAB extraction: a method particularly effective for extracting DNA from plant
tissues, using cetyltrimethylammonium bromide to remove interfering
components.
Why do we need different protocols for
DNA Isolation ?

 Due to difference in the cell structure of organisms, we require

different protocols for the isolation of DNA.

 For example, the CTAB method is commonly used for the

isolation of DNA from plants due to the presence of cell wall.

 In comparison to plants , isolation from animal cells requires

lesser effort and different approaches to isolate DNA as animal
cells are not enveloped in a cell wall.
Protocol for Genomic DNA
Isolation for plants
 Isolation of Total Genomic DNA:
Collection of sample:

Fresh leaf samples of plants were used for DNA extraction process and collected

leaf samples were stored in deep freezer at -20°C to -56°C; for further investigation.

Table 1: Preparation of Stock solutions

Sl no Chemicals Quantity/litre

1 1M Tris HCl 121.1g

2 5M NaCl 292.2g

3 0.5M EDTA 186.2g

Table 2: Preparation of Isolation Buffer:

Sl No. Reagents Quantity/100ml

1 CTAB – 2% 2g
2 100 mM Tris HCl 10ml
3 1.4M NaCl 28ml
4 20mM EDTA 4ml
5 1% Polyvinylpyrolidone (PVP) 1g

6 0.2% β-mercaptoethanol 0.2ml

 Modified protocol for DNA
extraction
Grind leaves
Transfer the Incubate at Add equal Transfer the Add double Incubate at
Take leaf leaf powder to 60°C for 60 volume of C:I
into a fine upper the volume -20℃ for
sample powder using an Oakridge minutes with (24:1) and mix aqueous of pre- overnight.
liquid nitrogen tube intermittent by slight
and a pre- layer to a chilled
chilled mortar containing pre- swirling every inversion. new fresh isopropanol.
and pestle. warmed 15 minutes by Centrifuge at tube using
extraction inverting the 10,000 rpm for wide-bore
buffer. tube. 30 min. pipette tips.
& vortex.
Centrifuge the Add an equal Centrifuge Centrifuge
Wash the DNA Dissolve the The samples Dry the
sample at pellet with DNA pellet in were treated volume of at 12,000 the sample at
10,000 rpm pellet and
70% ethanol. 1ml TE buffer Purification with RNase Phenol, rpm for 15 10,000 rpm add TE
for 15 min. to Dry the pellet A. Incubate Chloroform,
and transfer steps minutes. for 15 buffer. Store
precipitate at room in fresh the sample
DNA. Discard temperature. at 37 °C for and Isoamyl Transfer minutes. at 20 °C.
Eppendorf alcohol
the tube.
30 minutes upper layer. Discard the
in a water (25:24:1), and Add double
supernatant. supernatant,
bath.
mix by slight volume of wash the
inversion. chilled DNA pellet
ethanol and with 70%
store at -20 ethanol.
°C for 1
hour.
 Quantitative and qualitative
assessment of extracted
Samples
The quantity of extracted DNA samples will be verified spectrophotometrically using nanodrop and
quality by agarose gel electrophoresis. The concentration, purity, and absorbance were measured at
260/280 nm for the detection of contaminants such as proteins, salts, and polysaccharides, 1.6 to 1.8
absorbance showed the isolated DNA is free from contaminants. Stock solution of DNA dilute to make
10 μm concentration working solution for genomic test with 7μl of DNA and 3μl of 5x green buffer.
Using 1% agarose gel, electrophoresis will be performed with 1x TBE buffer containing 1µg/ml of
ethidium bromide (EtBr) at a constant voltage of 100 V for 30 min. The DNA bands were visualized
using the Gel Documentation System or UV transilluminator.
Protocol for Genomic DNA Isolation
from Animal Tissue OR BLOOD
 Material

 Lysis buffer (TE)

 Proteinase K
 Phenol-chloroform isoamyl alcohol (PCI)
 SDS 10%
 TNE buffer
 Isopropanol
 Ice cold 95-100% ethanol
 Ultrapure (DNA- & DNase-free) water
 Lab equipment needed:
 Pippetes,
 1.5mL sterile microcentrifuge tubes or 15, 50 mL Falcons,
 Racks,
 Tips
 Vortex
 Freezer
 Centrifuge

Blood Sample
Blood collection in anticoagulant i.e. Ethylenediamide tetra-acetic
acid (0.5 M EDTA) containing tube 1.5 mL eppendorf or 15 mL
Falcon tube

Storage of Blood Samples

Field blood samples should be place on ice immediately after their
collection store in freezer at -20°C before DNA extraction
 Steps in Organic and Inorganic DNA
Extraction
1- Lysis of Red Blood Cells, RBC

2- Digestion step (Lysis of White blood cells, WBC)

3- Phase Separation step (Extraction of Protein)

Organic DNA Extraction: PCI

Inorganic DNA Extraction: 6M NaCl

4- DNA Precipitation

5- Washing with ice cold Ethanol

6- Dilute the pellet

1. Lysis of Red Blood Cells, RBC

1. Lysis of red blood cells

2. Add 800 L of Tris EDTA buffer (Tris HCl 10mM, EDTA 2mM) in
200 ml of the blood. Mixed by inverting several times.

3. Centrifuge at 5000 rpm for 10 min.

4. Discard the supernatant.

5. Break the pellet formed at the bottom of the eppendorf tube by

tapping it gently. Add 1 mL TE buffer and mixed it gently.

6. Centrifuge at 5000 rpm for 10 min. this step may be repeated until
pellet becomes light pink.
2. Digestion step (Lysis of White blood cells, WBC)

Pellet obtained after lysis of RBCs re-suspended in

 400 L Buffer TNE (Tris HCL 10mM, EDTA 2mM, NaCl 400mM),

 200 L 10% SDS

 50 L Proteinase K

Homogenize the tube with gentle rotation

Samples incubate overnight in 58 °C in shaker water bath.

Next Day
3. Phase Separation step (Extraction of Protein)

In this step, we can remove the digested protein through

 6M NaCl (Inorganic Method) or
 Phenol-chloroform isoamyl alcohol (PCI, in ratio 25:24:1 respectively)
(Organic Method).
DNA released into solution is extracted with PCI to remove proteinaceous
materials.
Add equal volume of phenol-chloroform-isoamyl (PCI) alcohol. Mix
gently for 2 min and centrifuge for 10 minutes at 10,000 rpm.

Carefully remove the top (aqueous) phase

containing the DNA using a 1000- L pipette
transfer to a new tube.
 4. DNA Precipitation
 Precipitate the DNA with absolute isopropanol and inverted the tubes
gently till DNA threads became visible and then left the tubes at room
temperature for 10 minutes.

 Centrifuge at 8000 rpm for 10 minutes and discarded the supernatant

carefully and white pellet of DNA may visible at the bottom of the tube.

5. Washing with ice cold Ethanol

 Wash DNA pellet with 1 mL of 70-100% ethanol, break and mix the
pellet

 Then centrifuge at 8000 rpm for 10 minutes and discarded the

supernatant carefully

 Air dried the DNA pellet at room temperature for at least 2 hours
 6. Dilute the pellet

 Add 50-100 L of low T.E. (Tris HCl 10 mL, EDTA 0.2mM)

or DEPC water

 Place the tubes in a shaking water bath at 70°C for one hour
so that nucleases were inactivated. Finally DNA will store at
–20oC.
 APPLICATION OF DNA ISOLATION

Scientific Research:

Genetic studies:
DNA isolation allows researchers to study the structure and function of genes, identify genetic mutations, and
investigate the genetic basis of various traits and diseases.

Genome sequencing:
Isolated DNA is essential for sequencing genomes, which provides a complete map of an organism's genetic
material.

Evolutionary studies:
DNA analysis helps researchers understand the evolutionary relationships between different species.

Medical Diagnostics:
Genetic testing:
DNA isolation is used for a variety of genetic tests, including prenatal testing, carrier testing, and diagnostic tests
for inherited diseases.

Cancer diagnosis:
DNA analysis can help identify cancer-related mutations and develop targeted therapies.

Drug development:
Isolated DNA is used in the development of new drugs and therapies, particularly in the field of genetic
engineering.
Forensic Science:
DNA fingerprinting:
DNA isolation is essential for creating DNA profiles, which can be used to identify individuals and
solve crimes.

Paternity testing:
DNA analysis can determine the biological relationship between individuals, such as in paternity cases.

Other Applications:
Plant biology:
DNA isolation is used to study plant genetics, identify specific traits, and develop improved crop
varieties.

Environmental science:
DNA can be used to identify and detect bacteria and viruses in the environment.

Recombinant DNA technology:

Isolated DNA is used to create recombinant DNA, which involves combining DNA from different
sources to produce new traits.