ÜSKÜDAR UNIVERSITY

FACULTY OF ENGINEERING AND NATURAL

SCIENCES
DEPARTMENT OF BIOENGINEERING

BEN328 GENETIC ENGINEERING LABORATORY

2023-2024 SPRING SEMESTER

 EXPERIMENT 1: DEOXYRIBONUCLEIC ACID (DNA) EXTRACTION FROM
SOME FRUITS AND VEGETABLES

Introduction
➢ DNA
Deoxyribonucleic acid (DNA) is a molecule that encodes genetic instructions. The instructions
coded into the DNA guide the development and functioning of all known living organisms and
many viruses. Similar to the way a builder uses a blueprint to construct a house, cells use DNA
to construct an organism. DNA is therefore often considered the “blueprint for life.”
DNA instructions are divided into segments called genes. All organisms have genes that
determine various biological traits. Some are immediately visible, such as eye color or hair
color, and some are not, such as blood type or musical talent. Each gene provides the
information for making a protein that carries out a specific function in the cell. The world
depends on plants and animals for food, clothing, shelter and fuel every day. These plants and
animals are comprised of cells that rely on DNA for their development, functioning and
reproduction. In agriculture, scientists evaluate the DNA to find genes that code for specific
traits, such as disease resistance, drought tolerance and higher yield, to improve the plants.
These improvements benefit you as consumers when you purchase products with improved
nutrition, larger size and better taste.

➢ DNA Extraction
DNA extraction is a method to purify DNA by using physical and/or chemical methods from a
sample separating DNA from cell membranes, proteins, and other cellular components.
Extraction of nucleic acids is the starting point in any molecular biology study and hence is
considered as a crucial process. A Swiss physician Friedrich Miescher had performed the first
crude extraction of DNA in 1869. He had accidentally purified DNA from the nucleus while
investigating proteins from leukocytes and found that the property of this substance was
fundamentally different from proteins, hence coined the term “nuclein”. DNA can be extracted
from sources as diverse as clinical samples such as fine needle aspirates of body fluids and
biopsy samples; forensic samples such as dried blood spots and fingerprints; soil, plant and
animal tissue, insects, protozoa, bacteria, and yeast. The extraction of intact, high-molecular-
mass genomic DNA is essential for many applications including long range PCR, endonuclease
restriction digestion, southern blot analysis, and genomic library construction etc.

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Manual methods as well as commercially available kits are used for DNA extraction. Since the
first DNA extraction performed by Friedrich Miescher in 1869, scientists have made
extraordinary progress in designing extraction methods that are more reliable, easier and faster
to perform, more cost-effective and produce a higher yield. DNA extraction involves lysing the
cells and solubilizing DNA, which is followed by chemical or enzymatic methods to remove
macromolecules, lipids, RNA, or proteins.
The principal behind DNA extraction consists of the following steps:
1. Disruption of cytoplasmic and nuclear membranes;
2. Separation and purification of DNA from other components of the cell lysate such as
lipids, proteins, and other nucleic acids;
3. Concentration and purification of DNA
When choosing a suitable method for DNA extraction, it is crucial to ensure the quality and
quantity of the isolated DNA to carry out the intended applications. Other factors that should
be taken into consideration to optimize the DNA extraction method include the time, cost,
potential toxicities, yield, laboratory equipment and expertise requirements, as well as the
required sample amount for the protocol. Therefore, a good extraction procedure for the
isolation of DNA should yield adequate and intact DNA of reasonable purity. However,
procedure should be simple, inexpensive, and quick.
Principle of the experiment: Scientists can buy ready-to-use DNA extraction kits. These kits
help extract DNA from particular cell types or sample types. However, they can be expensive
to use routinely, so many laboratories have their own methods for DNA extraction. In the
experiment, our principle is extraction of DNA from plant cells and learning extraction
techniques.
Method
DNA extraction is a fairly simple process. The first step is to create the lysis buffer. Lysis is a
Greek word that means to break open. The lysis buffer (water, detergent and salt) breaks open
the cells by destroying the fatty membranes that enclose the cells as well as the nuclear
membranes within the cells. DNA is released into the solution. The salt in the lysis buffer strips
away proteins associated with the DNA molecules, and alcohol helps the DNA precipitate out
of the solution. The colder the alcohol, the more DNA will precipitate.
-Making Lysis Buffer (DNA Extraction Buffer)
1. Add 5 g of salt (NaCl) to the empty 50 mL lysis buffer tube.
2. Add 45 mL of water (H2O) and 5 mL of clear liquid detergent to the lysis buffer tube.
3. Place the cap securely on the tube and mix gently by swirling or inverting the tube.
-Making Tissue Lysate
1. Choose a fruit or vegetable as a tissue sample (Kiwi, mango and strawberry have been
found to yield the most DNA. Banana, onion, apple, and broccoli are also good
samples.)
2. Peel any tough skin and take out large seeds from the selected tissue sample. Cut the
sample into small pieces.

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 3. Place some amount of the selected sample in a stomacher bag, a sturdy plastic bag or a
laboratory porcelain mortar.
4. If you use a stomacher or plastic bag, remove all air from the bag and seal it well.
5. Mash the sample in the bag/mortar to a pulp.
6. Add the entire lysis buffer on to the pulp.
7. If you use a stomacher or plastic bag, remove the air and seal the bag again.
8. Mix the pulp with the added lysis buffer.
9. The required fruit/vegetable lysate have been made. The lysate consists of lysed
fruit/vegetable cells meaning that the cells have been “broken open.”
- Filtration of the Fruit/Vegetable Lysate
1. Place a filter paper into a funnel, and place the funnel into a beaker.
2. Pour the lysate from the bag/mortar into the filter paper. The liquid that drips from the
funnel into the beaker is called as filtrate. If filtrate is moving too slowly through the
filter paper, as can be the case with bananas, the filtrate can dilute with distilled water.
3. The filtrate includes the DNA.
4. Place the filtrate on ice for about 5 minutes.
- Visualization of the DNA
1. Remove the funnel from the beaker.
2. Add 25 mL of ice-cold ethanol or 91% isopropyl alcohol slowly to the beaker by pouring
down the side.
3. Observe the interface between the ethanol (or 91% isopropyl alcohol) and lysate layer.
The DNA will begin to precipitate out of the lysate.
4. Collect the extracted DNA using a glass rod.