2

Aim of the experiment:

The aim of this experiment is to understand and perform three procedures DNA
isolation, PCR and agarose gel electrophoresis.

Introduction:

DNA isolation:

Deoxyribonucleic acid (DNA ) isolation is an extraction process of DNA from

various sources. simply it purifies DNA by using physical or chemical methods
from a sample separating DNA from proteins, lipids, RNA, cell membranes, and
other cellular components. Friedrich Miescher in 1869 did DNA isolation for the
first time. For genetic analysis, which is used for science, medical, or forensic
purposes, DNA isolation is needed. DNA can be extracted from a number of
different sources. It can be separated from any living or dead organism. for
example: hair, sperm, bones, nails, tissues, blood stains, saliva, buccal (cheek)
swabs, urine, sample cards, bacteria, animal tissues, or plants are some of the
most common sources for DNA isolation. To do DNA isolation there are three
main steps should be done, which are lysis, precipitation and purification. The
first step to extract DNA from a tissue/cell, the cell
membranes have to be disrupted. The nucleus and
the cell are torn open during lysis, releasing DNA. In
the lysis buffer EDTA removes the ions which are
important for cell membrane maintenance and Fig 4.1: cell suspension

detergent SDS removes the lipids of the cell membranes are used to dissolve
 3

cellular proteins and free DNA. Precipitation is the next step, which separates the
freed DNA from the cellular debris. Sodium (Na+) ions are used to neutralize any
negative charge in DNA molecules, rendering them less water soluble and more
stable. The addition of alcohol (e.g., isopropanol or ethanol) causes DNA to
precipitate from the aqueous solution since it does not dissolve in alcohol. Last
step is Purification which is the method of rinsing DNA with alcohol after it has
been separated from an aqueous solution. Purification gets rid of any leftover
cellular debris or unnecessary material. After DNA has been fully purified, it is
normally dissolved in water once more for storage and handling. After centrifuge,
DNA pellet will be visible on the bottom of the tube. Once DNA is isolated it can
be used for molecular analysis such as PCR, electrophoresis, sequencing,
fingerprinting, and cloning.

Fig 4.2: DNA extraction steps

https://www.encyclopedia.com/science-and-technology/biology-and-genetics/genetics-and-
genetic-engineering/dna-isolation-methods

https://www.aatbio.com/resources/faq-frequently-asked-questions/What-are-the-steps-of-
DNA-extraction
 4

Polymerase Chain Reaction (PCR):

The polymerase chain reaction (PCR) is a technique that is widely used in the lab
to make millions of copies (amplify) of a particular section of DNA. It was first
developed in the 1980s by the American biochemist Kary Mullis. A small amount
of DNA can be used to produce thousands to millions of copies of a specific
section of DNA using PCR. Many techniques used in genetic testing and study,
such as the analysis of ancient DNA samples and the detection of infectious
agents, depend on it. PCR is now is a fundamental technique that is a widely used
and often indispensable tool in medical laboratory research, with applications
ranging from scientific research to criminal forensics. The use of PCR in medical
application are like, genetic testing for presence of genetic disease mutations. Eg:
hemoglobinopathies, cystic fibrosis. Also, helps to monitor the gene in gene
therapy and many more. Using PCR in forensic applications, can be used as a tool
in genetic fingerprinting. This technology can distinguish one person from millions
of others in the following situations: crime scene analysis, ruling out suspects
during police investigations, paternity testing even when only a limited number of
specimens are available ( stains of blood, semen, hair etc). There are many types
of PCR like conventional PCR, real-time PCR, quantitative real time PCR (Q-RT
PCR), Multiplex PCR and many more. Five basic ‘ingredients' are required to set
up a PCR. These are; the DNA template that will be replicated, primers which
short stretches of DNA that start the PCR reaction and are designed to bind to
both sides of the DNA segment that will be replicated, DNA nucleotide bases(
(also known as dNTPs)
which are the building
blocks of DNA (A, C, G,
and T) are needed to
Fig 4.3: DNA template
create the new strand
of DNA, Taq polymerase enzyme which is the enzyme that catalyzes the
production of DNA copies. Since modern PCR involves high temperature steps, a
heat-resistant DNA polymerase is used, lastly buffer which keeps the solution at
an optimal pH for the PCR reaction to occur. The entire cycling process of PCR is
automated on thermal cycling machine. This process involves repeatedly heating
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and cooling reactants to allow for temperature-dependent reactions such as DNA

melting and enzyme-driven DNA replication. There are three main stages of PCR;
first is denaturing which is the process of separating the double-stranded
template DNA into two single strands by heating it. Second stage is annealing
which is the process of lowering the temperature to enable the DNA primers to
bind to the template DNA. Third stage is extending, which occurs when the
temperature is increased and then the Taq polymerase enzyme creates a new
strand of DNA. So, simply to amplify a segment of DNA using PCR, the sample is
heated first, causing the DNA to denature, or split into two single-stranded
fragments. Then, using the original strands as bases, an enzyme called "Taq
polymerase" synthesizes - or builds - two new strands of DNA. This process causes
the original DNA to be duplicated, with one old and one new strand of DNA in
each of the new molecules. After that, each of these strands can be used to make
two more copies, and so on. The cycle denaturing and synthesizing new DNA is
replicated 30 or 40 times, resulting in over one billion exact copies of the original
DNA segment. once the PCR has been completed, the quantity and size of the
DNA fragments generated can be tested using electrophoresis.
https://www.yourgenome.org/facts/what-is-pcr-polymerase-chain-reaction
https://www.genome.gov/about-genomics/fact-sheets/Polymerase-Chain-Reaction-Fact-Sheet
https://www.khanacademy.org/science/ap-biology/gene-expression-and-
regulation/biotechnology/a/polymerase-chain-reaction-pcr

Fig4.4: Stages of PCR

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Agarose Gel Electrophoresis:

Nucleic acid fragments are identified, quantified, and purified using gel
electrophoresis, a common lab technique. Gel electrophoresis is a technique for
separating molecules based on how quickly they pass through a gel under the
influence of an electric field.it separates charged molecules on the basis of size
and charge. Agarose electrophoresis is a common method for separating,
identifying, and purifying DNA
fragments. The method is simple to
use, quick to implement, and capable
of resolving DNA fragments that are
difficult to separate using other
techniques. The nucleic acids can be
separated as whole chromosomes or
as fragments. Macromolecules are Fig 4.5: Gel electrophoresis
forced to migrate through pores during electrophoresis, and their rate of
migration through the electric field is determined by the following factors: the
strength of the field, the molecule's size and shape, the samples' relative
hydrophobicity, the ionic strength, and the temperature of the buffer in which the
molecules are moving. Samples are loaded into wells of an agarose gel and
exposed to an electric field, which causes negatively charged nucleic acids to
migrate to the positive electrode. Shorter DNA fragments will migrate faster,
while the longest fragments will stay closer to the gel's origin, resulting in size
separation. Agarose is a linear polymer extracted from seaweed, it Forms a
porous matrix as it gels and shifts from random coil in solution to structure in
which chains are bundled into double helices. The materials needed to perform
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agarose gel electrophoresis are electrophoresis chamber, gel casting trays, a

comb, agarose gel, buffer, staining agent (dye), DNA ladder and a sample to be
separate. The five main steps to perform gel electrophoresis are: pouring the gel,
preparing the sample, loading the gel, running the gel and last staining the gel.
The agarose gel is prepared by combining agarose powder and a buffer solution.
Buffers are used to prevent the pH from changing by reacting with the H+ or OH-
products. Most common buffer used is called TRIS. To prepare a sample 6X
loading buffer, bromophenol blue (for color) and glycerol (for weight) are used.
This increases the density of the samples, allowing them to sink into the gel wells,
which allows them to be seen when loading onto the gel. To make DNA
fragments visible after electrophoresis, the DNA is stained with ethidium
bromide. Ethidium bromide is a fluorescent dye that intercalates between
nucleotide bases, making it easy to identify DNA fragments in gels. The DNA
ladder is made up of known DNA sizes that are used to figure out how big an
unknown DNA sample is. The DNA fragments glow when a gel is stained with a
DNA-binding dye and exposed to UV light, allowing us to see the DNA present at
various positions along the length of the gel. Each band comprises a large number
of identical-sized DNA fragments that have all traveled together to
the same location. You can estimate the sizes of bands in a sample
by comparing them to the DNA ladder. simply, agarose gel
electrophoresis is a technique for separating DNA molecules
that is widely used in DNA manipulation techniques or studies
involving identifying individuals based on their specific DNA
sequence.
https://www.yourgenome.org/facts/what-is-gel-electrophoresis

Fig 4.6: DNA ladder

 8

https://www.khanacademy.org/science/ap-biology/gene-expression-and-
regulation/biotechnology/a/gel-electrophoresis

Procedure 1: DNA Isolation

Materials:
• Jurkat Cell Suspension
• Collection Tube
• Lysis Solution
• Resuspension Solution
• Centrifuge
• Micropipette
• Wash Solution
• 96% Ethanol
• Thermomixer
• Tissue paper
• Column Preparation Solution
• Spin Column

procedure1:
1. cell suspension is centrifuged at 300 grams for 5 minutes. After
centrifugation culture media is discarded using a tissue paper.
2. Using a micropipette 200 ul of resuspension solution is added to the pellet
and shaken up and down till the solution is distributed.
3. Also, 200 ul of lysis solution is added to the pellet using a micropipette and
shaken gently.
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4. Then, the sample is placed in a thermomixer and incubated at 70°C for 10

min and then taken out.
5. After that, using a micropipette 500 ul of column preparation solution is
poured on top of the column and centrifuged at 12000 gram for 1 minute.
when it’s done flow-through liquid is discarded.
6. To precipitate the DNA, 200 ul of 96% ethanol is added to the sample using
the micropipette and shaken up to ensure an even mix.
7. Then two filters are used, one of the filters is used to pass the sample while
the other filter is used as a counterbalance. Both column filters are
centrifuged at 6000 g for 1 minute and then flow-through liquid is
discarded.
8. Using a micropipette 500 ul of wash solution is added to the column that
will be used. For the balance one, its adjusted using the addition of water.
9. Both columns are centrifuged for 1 minute and flow-through liquid is
discarded again.
10.After that, the filter is removed and changed to a new collection tube and
the sample name and date is written on it for the PCR use.
11.To retain the DNA molecules, using a micropipette 100 ul of water is
pipetted carefully on top of the filter. Same procedure is done for the
balance.
12.Lastly, the columns are centrifuged again, and the filter is discarded from
the sample after centrifugation. Now the DNA is eluted.

Procedure 2: PCR
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Materials:
• Thermocycler
• PCR Tubes
• Template DNA ( BTK gene )
• PCR mix
• Primer Set

Procedure 2:
1. Using a micropipette 4 ul of DNA is added to PCR tube and also 4 ul of
water is added to another PCR tube as a negative control.
2. Then, 1 ul of primer set starting from A is added to a PCR tube and another
same sample is taken and added to a negative control tube. So, in total 2
primer sets are used.
3. After that, a 20 ul of PCR mixture is added to the DNA PCR tube and the
same for the negative control PCR tube. So, in total 40 ul of PCR mixture is
used.
4. These steps are repeated 18 times from A to T tubes as the BTK gene has 19
exons.
5. Finally, the PCR tubes are placed into the thermocycler to run the device.

Procedure 3: Agarose Gel Electrophoresis

Materials:
• Agarose powder
• Cassette and combs
 11

• Analytical balance
• TEA Buffer
• Microwave
• Erlenmeyer Flask
• Ethidium Bromide
• UV Machine
• Micropipette

Procedure3:
1. To do to 2% gel, 3 grams of agarose powder are weighed using an analytical
balance and placed in an Erlenmeyer flask and 150ml of TEA buffer were
added to it.
2. The Erlenmeyer flask is microwaved till the agarose begins to boil, as like
this the agarose is dissolved and then it’s taken out to cool and placed on a
plate.
3. Then, 3 drops of ethidium bromide are added to the flask to stain the gel.
4. After that, the cassette is assembled, and two combs are mounted on top
of it.
5. Then, the agarose gel is poured on the cassete and its left till the gel
solidifies.
6. Using a micropipette, the samples from the previous procedure are loaded
to each well and 5 ul of DNA ladder is loaded in the first lane.
7. Then, the gel is placed in the tank and the lid is placed on and connected to
the supper supply.
8. Lastly, DNA is visualized using a UV light.
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Results:

Figure 4.7: DNA fragments

The approximate size of each numbered DNA:

1 : 450 8: 550 15: 500

2: 550 9: 300 16: 200
3: 350 10: 250 17: 390
4: 250 11: 150 18: 300
5: 400 12: 250 19: 850
6: 400 13: 350
7: 300 14: 400
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Discussion:
In this experiment three parts were done to visualize the DNA of the Jurkat cell
line DNA isolation, PCR and Agarose Gel Electrophoresis. In the first part DNA is
isolated by three main steps. first cell suspension is centrifuged to separate the
DNA from the rest of the cell. Second step resuspension solution is added to the
pellet so its homogenized then the lysis step is done by adding a buffer lysis
solution so that the DNA can be extracted. Third main step, DNA is precipitated by
ethanol so that DNA strand aggregate together. Also, the DNA is water soluble
therefore it cannot dissolve in substance like alcohol, so ethanol is used. the
second and third part of the experiment were related to each other. In the second
part BTK gene was amplified using the PCR. The main step to do PCR is to prepare
the PCR mixture which contains water, buffer, DNA templates, Taq DNA
polymerase, MgCl2 and dNTPs. Also, negative control was done to ensure that no
contaminating nucleic acid was added into samples during the sample processing
process. The negative control does not receive any treatment. So, it does not
show any change during an experiment that’s why it’s used to control unknown
variables during an experiment, and it compared with the main one. Running PCR
usually takes a lot of time, but it depends on each one but one its done, the DNA
can be detected using gel electrophoresis which is the last part of the experiment.
The most common method for detecting PCR products is gel electrophoresis. Gel
electrophoresis is a technique used to separate DNA fragments based on their
size making it easier to differentiate and visualize them. In this experiment
agarose gel electrophoresis was done and one of the main things to prepare an
agarose gel electrophoresis is to do the gel. In the experiment 2% gel was done by
mixing agarose powder with TEA buffer and then it was microwaved till it was
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dissolved. So, it will end up looking crystal clear. Ethidium bromide stain was
added to the gel as its an intercalating agent, so it stains the DNA. Later on, the
gel is poured to the cassete and two combs are assembled to it. The DNA ladder is
loaded, and the sample is loaded and ready to run. When the ethidium bromide is
exposed to the UV light it intensifies 20 nearly times and fluoresces orange when
it’s bound to the DNA. In agarose gel electrophoresis, a DNA ladder is a solution of
DNA molecules of various lengths. It's used as a standard for estimating the size of
unknown DNA molecules isolated on a gel based on their mobility in an electrical
environment. The DNA ladder consists of 10 fragments from 100 to 1000 bp. To
end up, agarose gel electrophoresis is most common method to detect PCR
products as it is easy to visualize and use.

References:

• https://www.encyclopedia.com/science-and-technology/biology-and-
genetics/genetics-and-genetic-engineering/dna-isolation-methods
• https://www.aatbio.com/resources/faq-frequently-asked-
questions/What-are-the-steps-of-DNA-extraction
• https://www.yourgenome.org/facts/what-is-pcr-polymerase-chain-
reaction
• https://www.genome.gov/about-genomics/fact-sheets/Polymerase-
Chain-Reaction-Fact-Sheet
• https://www.khanacademy.org/science/ap-biology/gene-expression-
and-regulation/biotechnology/a/polymerase-chain-reaction-pcr
• https://www.yourgenome.org/facts/what-is-gel-electrophoresis
• https://www.khanacademy.org/science/ap-biology/gene-expression-
and-regulation/biotechnology/a/gel-electrophoresis