Bio 14 Integrative Biology Laboratory

EXERCISE 5
GENOMIC DNA ISOLATION

INTRODUCTION

Technical breakthroughs through the years have created a great impact on different aspects of cell biology
by facilitating the study of cells and their macromolecules including DNA. In particular, the development of
the recombinant DNA technology has ushered in a new and powerful way of manipulating the DNA, which
in turn, allowed obtaining inferences on gene regulation, protein function, organismic diversity and
phylogenetic relationship among others.

Many molecular studies require the isolation of DNA from various organisms. The DNA that constitutes the
totality of genetic information belonging to a cell or organism is referred to as genomic DNA. Genomic DNA
can be directly extracted from a sample and is often differentiated from complementary DNA (cDNA)
prepared by reverse transcription from messenger RNA (mRNA).

Extraction of high-quality genomic DNA from an organism is a prerequisite to other DNA manipulation
techniques such as restriction digestion. Gene cloning, amplification via polymerase chain reaction (PCR),
Southern blotting, DNA sequencing, and other methods which are involved in the molecular characterization
of genes.

Essentially, DNA isolation requires the removal of other interfering substances in cells such as polysa ccharides,
lipids, proteins, and enzymes (specifically nucleases that degrade nucleic acids). Classical procedures include:
cell lysis often mediated by detergents, treatment with enzymes such as proteinase, use of chemicals that
inactivate nucleases, extraction with organic solvents, and ethanol precipitation.

Many protocols for DNA isolation have already been developed, and variations largely depend on the type
of specimen from which the DNA will be obtained, which could be any microbe, plant, animal or other
organism. These different organisms may contain substances that can interfere with the isolation process or
even degrade DNA and as such will require specific modifications in the isolation process. The cell wall in
bacteria and in plants present difficulty in DNA isolation, while secondary plant products like phenolic
terpenoids cause DNA degradation if not removed after cell lysis.

Although protocols vary, the goal of any DNA isolation procedure is to extract high-quality DNA that can be
used for various applications. In particular, the DNA obtained should preferably be of a a) high molecular
weight (i.e. with minimal shearing), b) high purity as indicated by a 260/280 absorbance ratio of 1.8 – 2.0, and
c) high yield with respect to the amount of tissue used.

OBJECTIVES

1. To discuss the significance of DNA isolation in cell and molecular biology experiments;
2. To explain the principles involved in the methods employed during isolation of genomic DNA; and
3. To differentiate some protocols of DNA isolation.
Exercise 5. Genomic DNA Isolation

Sample Activity for Genomic DNA Isolation

MATERIALS

CTAB Extraction Buffer Tris-EDTA (TE) Buffer

Chloroform : isoamyl alcohol (24:1) Sterile distilled water
Isopropanol
70 % Ethanol

PROCEDURE

1. Collect young leaves from a plant and wash with distilled water. Blot dry with tissue paper.
2. Pulverize 1 g of leaf tissue in liquid nitrogen in a pre-chilled mortar and pestle. Add 3 mL 2x CTAB buffer
and mix gently by swirling. Note: In the absence of liquid nitrogen, directly grind the tissue in CTAB
buffer with the mortar placed in ice bath.
3. Transfer the homogenate into fresh, sterile mirocentrifuge tubes using a sterile plastic dropper, placing
about 1 mL per tube. For samples that were not homogenized in liquid nitrogen, pipet out only the liquid
part of the homogenate.
4. Incubate the mixture at 60-65°C for 30 min in a water bath.
5. Centrifuge the tubes at 10, 000 rpm for 15 min at 4°C to allow the cell debris to sediment. Collect the
supernatant and transfer to new microcentrifuge tubes taking note of the volume transferred.
6. To each tube, add an equal volume of chloroform:isoamyl alcohol (24:1). Mix gently for one minute by
tube inversion and centrifuge at 10, 000 rpm for 15 min at 4°C.
7. After centrifugation, two liquid phases will form. Avoiding the interface, pipette out the aqueous (top)
phase into a new microcentrifuge tube taking note of its volume. Add an equal amount of ice-cold
isopropanol. Mix gently to precipitate the nucleic acids. If no precipitate is visible, place at -20°C for 20
min or longer.
8. Centrifuge ate 13, 000 rpm for 15 min at 4°C. Gently pour off as much of the supernatant as possible
without losing pellet.
9. Add 1 mL ice cold 70% ethanol and flick the pellet of the wall of the microcentrifuge tube to wash it.
Generally, nucleic acids will become whiter at this step. Centrifuge ate 13, 000 rpm for 2 min at 4°C.
Repeat this step two to three times.
10. Pour off the washing solution and dry the resulting DNA pellet for about 5 min by inverting the tube on
a paper towel in a fume hood. Add 50 µL of TE buffer and resuspend the pellet by gently flicking the
bottom of the tube.
11. Check the purity and/ or concentration of the genomic DNA sample through a spectrophotometer or by
comparison with standard concentrations through gel electrophoresis. It maybe necessary to further
purify the DNA by including more than one chloroform extraction or by including a final phenol
extraction. The extracted genomic DNA may be stored at -20°C for future use.

ACTIVITY

Virtual lab learning

QUESTIONS

1. Explain the relevance of DNA isolation in cell and molecular biology experiments.
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Exercise 5. Genomic DNA Isolation

2. Provide the rationale for the use of the following reagents in DNA isolation:
3. Give the significance of the following steps in DNA isolation:
4. Research on a protocol for isolating DNA (aside from a plan tissue) from an organism and outline the
steps (either number the steps or present in a flowchart).
5. Explain the principle involved in the use of the 260/280 absorbance ratio for determining the purity
of a given DNA sample.

References

Reamillo, MCS, Mendoza JC, Diaz MGQ, Manuel MCC. 2009.Cell Biology Laboratory Manual 6th ed. Genetics and
Molecular Biology Division, Institute of Biological Sciences, College of Arts and Sciences, University of the Philippines
Los Baños

The Best of DNA and Genome Science. Cold Spring Laboratory, DNA Learning Center.

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