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Lab 4 Assignment - Abdullah Rashid (1)

The experiment aimed to extract and amplify DNA from cheek cells, focusing on the role of NaOH in cell lysis and the components of the Polymerase Chain Reaction (PCR). Cheek cells were lysed using NaOH to release DNA, which was then subjected to PCR involving Taq DNA polymerase and primers to replicate the DNA. The process included denaturation, annealing, and extension steps, ultimately producing millions of copies of the target DNA sequence.

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4 views

Lab 4 Assignment - Abdullah Rashid (1)

The experiment aimed to extract and amplify DNA from cheek cells, focusing on the role of NaOH in cell lysis and the components of the Polymerase Chain Reaction (PCR). Cheek cells were lysed using NaOH to release DNA, which was then subjected to PCR involving Taq DNA polymerase and primers to replicate the DNA. The process included denaturation, annealing, and extension steps, ultimately producing millions of copies of the target DNA sequence.

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Analyzing DNA Isolation and polymerase Chain reaction in cheek cells

Abdullah Rashid

221950050

Lab Section: N17

TA: Samira Kabir

 The purpose of this experiment was to extract and amplify a DNA sample of a cheek
cell to understand the role of the different enzymes and reagents that were used in the
process. Our primary focus was to see how NaOH helps in the process of cell lysis,
along with examining the components of the Polymerase chain reaction, and the
functionality of Taq DNA polymerase in DNA replication during high temperatures.
As we conducted this experiment, we analyzed the different steps that were involved
in PCR, which provided a showcase of how DNA could be manipulated for the use of
different scientific applications.

 The moment we began the experiment, we first collected cheek cell samples by using
sterile cotton swabs. The cells were then placed in a lysis buffer solution that
contained NaOH. The reason we placed the cheek cell into NaOH solution was to
break open the cheek cell by denaturing proteins and disrupting the phospholipid
bilayer of the membrane. During this process the DNA was released into the solution
by breaking the hydrogens bonds between base pairs, this allowed the genetic material
to be accessed more easily. Without NaOH, the DNA would have remained inside the
cells, and we wouldn’t have been able to extract the DNA. After the cells were lysed,
the samples were then neutralized and placed in centrifuge device that would separate
the cell particles from the extracted DNA. The DNA that was in the supernatant was
carefully extracted and transferred to a new tube for PCR analysis

 Once the DNA sample had been extracted, we mixed it with the PCR reaction mix.
The PCR reaction mix had different substances inside, which were deoxynucleotide
triphosphates or dNTPs, along with Taq DNA polymerase and primers. The dNTPs
are the building blocks for new DNA strands to be formed, while the Taq DNA
polymerase synthesizes DNA and remains stable during high temperatures. The
primer is what allows DNA replication to begin, as the primer attaches to specific
areas on the DNA sequence. We transferred the reaction mix into PCR tubes and then
placed them into a thermal cycler, which controlled the amount of heat for the
reaction. This process had three steps involved. The first was denaturation, in which
two strands that are double bonded to each other become separated into single strands.
This step occurs at around 95°C. The second step involves annelation, in which
primers attach to the target sequences. This process happens around 55-65°C. The
third step involves extension, in which Taq DNA polymerase constructs new DNA
strands. This whole process is repeated around 30 times and doubles the amount of
DNA each time producing millions of copies of the target sequence.

 During the elongation phase, which best operates at a temperature of 70°C, Taq DNA
polymerase builds new strands by attaching nucleotides to where the primer is
located. This enzyme is used in this process because it withstands the high heat
without breaking apart. If we had increased the temperature into the hundred °C
range, the enzyme would have stopped functioning, and the PCR wouldn’t have
worked. Primers are short sequences of DNA that attach themselves to a specific area
on the DNA we want to copy. DNA polymerase cannot begin adding nucleotides on
its own, rather it uses a primer to start building a new DNA strand. The way DNA
polymerase adds nucleotides is that a primer acts as the starting point for on the
targeted sequence for nucleotides to be added from three prime end which contains a
hydroxyl group to the fifth prime end which contains phosphate group.

 If the primers were complementary to each other, then they would have been stuck
together rather than binding on the targeted DNA. This would have created something
called prime dimers, which would have interfered with the PCR reaction, in turn
leading to reduced amounts of DNA copied. In our experiment the primers worked
correctly, so this did not happen. The forward and reverse primer had a distinct
orientation that started from the third prime to the fifth prime end. The forward primer
had base A as the fifth prime end and base B as the third prime end. The reverse
prime had a base C for the third prime end, and base D for the fifth prime end. We
already knew that DNA polymerase can only add new nucleotides from the fifth
prime to the third prime direction, this made sure that the correct DNA sequence was
amplified.

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