DNA ISOLATION

Student READY In-house Skill Development Module

(Plant biotechnology)

Submitted To: Submitted by : Shraddha Singh

Dr. Vaishali B-5187/20
(Dept. of Agriculture Biotechnology)
DNA isolation

DNA isolation is a process that involves the extraction of DNA from cells or tissues.
There are various methods for DNA isolation, and the choice of method depends on the
source of the DNA and the downstream applications.

Chemicals and reagents used in DNA isolation and

their role

The process of DNA isolation involves several chemicals, reagents, and enzymes, each
playing a specific role in the extraction and purification of DNA. Here are some key
chemicals and reagents used in DNA isolation and their roles:

Cell Lysis Buffer:

Role: Breaks down cell membranes and nuclear envelopes, releasing cellular contents.
Components: Detergents (e.g., SDS), salts, and sometimes protease inhibitors.

Proteinase K:
Role: Digests proteins, including nucleases, and removes them from the DNA.
Principle: Enzymatic digestion of proteins.
Note: Often used in combination with cell lysis buffer during the initial steps.

RNase A (Ribonuclease A):

Role: Degrades RNA present in the sample.
Principle: Enzymatic digestion of RNA.
Note: Helps prevent contamination of DNA samples with RNA.

Phenol-Chloroform-Isoamyl Alcohol (PCI) Mixture:

Role: Extracts proteins and other contaminants from the DNA sample.
Principle: Organic solvents like phenol and chloroform separate different biomolecules
based on their solubility.
Note: Used in the phenol-chloroform extraction step.

Isopropanol or Ethanol:
Role: Precipitates DNA from the solution.
Principle: DNA is less soluble in alcohol, causing it to precipitate.
Note: Used for DNA precipitation after the removal of proteins.

Ethanol or Isopropanol Wash Buffer:

Role: Washes and purifies DNA.
Principle: Helps remove residual salts and contaminants.
Note: Used in the washing step after DNA precipitation.
TE Buffer (Tris-EDTA):
Role: Resuspends and stores DNA.
Principle: Provides a stable, low-ionic-strength environment for
DNA. Note: Used for the final resuspension of isolated DNA.

These chemicals are often used in combination and as part of a larger DNA isolation
protocol. The overall goal is to break open cells, remove proteins, degrade RNA, and
then precipitate and purify the DNA. The specific methods and reagents used may vary
based on the type of sample, the scale of the experiment, and the downstream
applications for the isolated DNA. Researchers may also use commercially available DNA
isolation kits that provide pre-optimized reagents and protocols for efficient and reliable
DNA extraction.
And these reagents are integral parts of commonly used DNA isolation protocols, such
as the phenol-chloroform extraction method or silica membrane-based purification kits.
Commercial DNA isolation kits often provide pre-packaged versions of these reagents,
simplifying the process and ensuring consistency. Researchers should follow specific
protocols and safety guidelines when working with these reagents to obtain high-quality
and uncontaminated DNA samples.

Enzymes commonly used in DNA isolation and

their roles

Enzymes play crucial roles in DNA isolation protocols by facilitating specific reactions
that help in the extraction, purification, and manipulation of DNA. Here are some
enzymes commonly used in DNA isolation and their roles:

Proteinase K:
Role: Digests proteins.
Purpose: Used in the initial stages to break down proteins in the cell lysate. This step is
crucial for removing proteins that could interfere with DNA isolation and downstream
applications.

RNase A (Ribonuclease A):

Role: Digests RNA.
Purpose: RNA is often co-purified with DNA during extraction. Adding RNase A ensures
the removal of RNA contaminants, preventing interference with downstream applications.

DNase (Deoxyribonuclease):
Role: Digests DNA.
Purpose: Used in specific situations to eliminate genomic DNA contamination,
especially when RNA isolation is the primary goal.
Restriction Enzymes:
Role: Cleave DNA at specific recognition sequences.
Purpose: Used in certain DNA isolation techniques or applications, such as restriction
enzyme digestion to analyze DNA fragments or prepare DNA for cloning.

DNA Ligase:
Role: Catalyzes the formation of phosphodiester bonds between DNA fragments.
Purpose: Used in DNA cloning or ligation reactions where DNA fragments are
joined together covalently.

Polymerase Chain Reaction (PCR) Enzymes (e.g., Taq Polymerase): Role:

Synthesizes complementary DNA strands.
Purpose: Used in PCR for amplifying specific DNA regions. PCR is often employed in DNA
isolation workflows for targeted DNA amplification.

Alkaline Phosphatase:
Role: Removes phosphate groups from DNA.
Purpose: Used in certain DNA modification and labeling procedures, as well as in cloning
to prevent self-ligation of vector DNA.

Exonuclease I:
Role: Removes nucleotides from the ends of DNA.
Purpose: Used in DNA library preparation and other applications where the removal of
unincorporated nucleotides is necessary.

Endonucleases (e.g., EcoRI):

Role: Cleaves DNA at specific internal sequences.
Purpose: Used in DNA manipulation, such as generating specific DNA fragments with
defined ends for cloning or analysis.

The choice of enzymes depends on the specific requirements of the DNA isolation
method and the downstream applications. Enzymes are critical for ensuring the
specificity and efficiency of various steps in the DNA isolation process. Researchers must
carefully follow the recommended protocols for enzyme usage to obtain high-quality and
uncontaminated DNA samples.
DNA Isolation Procedure

The procedure for DNA isolation can vary based on the sample type (e.g., cells,
tissues, blood, bacteria) and the specific method or kit chosen. Below is a general
overview of a common DNA isolation procedure using a phenol-chloroform
extraction method:

Materials and Reagents:

Cell or tissue
sample Cell lysis
buffer Proteinase K
RNase A
Phenol-Chloroform-Isoamyl Alcohol (PCI) mixture
Isopropanol or ethanol
Wash buffer
TE buffer (Tris-EDTA)

Equipment:
Microcentrifuge
tubes Centrifuge
Vortex mixer
Heat block or water
bath Microcentrifuge

Procedure:

Cell Lysis:
a. Collect the cells or tissue sample in a microcentrifuge tube.
b. Add an appropriate volume of cell lysis buffer to the sample.
c. Mix the sample thoroughly (vortex or pipette up and down).
d. Incubate the sample at a specific temperature for a designated time to allow cell lysis.

Protein Digestion:
e. Add Proteinase K to the lysed sample.
f. Mix the sample thoroughly.
g. Incubate the sample at a specific temperature to digest proteins (typically 37-55°C).

RNA Digestion:
h. Add RNase A to the sample.
i. Mix the sample thoroughly.
j. Incubate the sample at a specific temperature to digest RNA (typically 37°C).

Phenol-Chloroform Extraction:
k. Add an equal volume of PCI mixture to the sample.
l. Mix the sample thoroughly.
m. Centrifuge the sample to separate the aqueous (DNA-containing) phase from the
organic phase.
n. Transfer the aqueous phase to a new tube.

DNA Precipitation:
o. Add isopropanol or ethanol to the aqueous phase.
p. Mix the sample thoroughly.
q. Centrifuge the sample to pellet the DNA.
r. Discard the supernatant.

Washing:
s. Wash the DNA pellet with ethanol or isopropanol wash buffer.
t. Centrifuge the sample.
u. Discard the wash buffer.

Resuspension:
v. Allow the DNA pellet to air-dry briefly.
w. Resuspend the DNA pellet in TE buffer or another suitable buffer.
x. Incubate at a specific temperature to ensure complete resuspension.

Quantification and Quality Check:

y. Measure the concentration of the isolated DNA using a spectrophotometer.
z. Assess the purity of the DNA by determining the A260/A280 ratio.

Storage:
a. Store the isolated DNA at an appropriate temperature for future use.

This general protocol provides an overview of the steps involved in DNA isolation using
a phenol-chloroform extraction method. It's important to note that specific kits or
protocols may have variations in reagents and steps based on the sample type and
downstream applications. Always follow the manufacturer's instructions or established
laboratory protocols for the most accurate and reliable results.
Conclusion

In conclusion, the isolation of DNA is a fundamental process in molecular biology,

genetics, and various biotechnological applications. The procedure involves several critical
steps to extract, purify, and preserve DNA from biological samples. The key steps include
cell lysis to release cellular contents, protein digestion to remove proteins, RNA digestion
to eliminate RNA contaminants, phenol-chloroform extraction to separate DNA from
proteins, DNA precipitation for DNA recovery, washing steps for purification, and final
resuspension in a suitable buffer.

The success of DNA isolation is crucial for downstream applications such as PCR, DNA
sequencing, cloning, and genetic analysis. Researchers must choose appropriate methods
and reagents based on the nature of the sample and the specific goals of their
experiments. Enzymes like Proteinase K and RNase A play pivotal roles in breaking down
proteins and RNA, respectively, ensuring the purity of the isolated DNA.

It's essential to follow established protocols, safety guidelines, and manufacturer

instructions to obtain high-quality DNA samples. Additionally, advancements in
biotechnology have led to the development of commercial DNA isolation kits, simplifying
the process and improving reproducibility.

In summary, DNA isolation is a foundational technique that enables scientists to unlock

the genetic information within cells, paving the way for advancements in various fields
such as medicine, agriculture, and forensic science.
References

1. Dhaliwal A (2013) DNA Extraction and Purification. Materials and Methods p. 3.

2. Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, et al.
(2002) Molecular biology of the cell (4th edn). Garland Science, New York, USA.
3. Hardison R (2020) 2.5: B-Form, A-Form, and Z-Form of DNA.
4. Eun H (1996) Enzymes and Nucleic Acids. Enzymology Primer for
Recombinant DNA Technology, pp. 1-108