Allyson Mendoza
221700729
Section B
QUESTION 1: What is the purpose of adding sodium hydroxide (NaOH) to the cheek cell
pellet, after removing Phosphate Buffered saline, on p. 55 of the extraction protocol? [1
mark] Note: You might need to conduct an internet search to answer this question. The
answer must be in your own words.
The purpose of adding sodium hydroxide (NaOH) to the cheek cell pellet after removing the
Phosphate Buffered Saline (PBS) is to facilitate the lysis of the cells and release their
contents, particularly DNA. NaOH disrupts the cell membranes and nuclear membranes,
allowing the cellular components to be extracted. Additionally, it denatures the DNA by
breaking the hydrogen bonds between the strands, which can be beneficial for subsequent
steps in the extraction protocol.
QUESTION 2: The 2x PCR Master mix (listed on p. 56+57) has four main components.
Two of these are PCR Buffer and MCl. Based on your knowledge of DNA replication and
PCR, what are the other two components and what are their functions? [4 marks]
The 2x PCR Master Mix contains four main components:
1. PCR Buffer: This provides the optimal pH and ionic environment for the PCR
reaction, ensuring that all components function effectively.
2. MgCl₂ (Magnesium Chloride): This is a cofactor that is essential for the activity of
DNA polymerase. It stabilizes the DNA structure and is required for the enzyme to
catalyze the addition of nucleotides during DNA synthesis.
3. dNTPs (deoxynucleotide triphosphates): These are the building blocks of DNA,
consisting of adenine (A), thymine (T), cytosine (C), and guanine (G). They provide
the necessary nucleotides for synthesizing new DNA strands during amplification.
4. DNA Polymerase: This enzyme synthesizes new DNA strands by adding dNTPs
complementary to the template strand. In PCR, a heat-stable polymerase like Taq
polymerase is commonly used due to its ability to withstand high temperatures during
denaturation.
QUESTION 3: What is the key enzyme responsible for the elongation step of the PCR
reaction? Would an increase of the temperature of this step from 72°C to 105°C have an
impact on the PCR reaction?
Explain your answer [3 marks]
The key enzyme responsible for the elongation step of PCR is DNA polymerase, specifically
Taq polymerase in many protocols. If the temperature during this step is increased from 72°C
to 105°C, it would significantly impact the PCR reaction:
1. Denaturation of Polymerase: While Taq polymerase is heat-stable, temperatures above
its optimal range can lead to denaturation of the enzyme, rendering it inactive and
unable to synthesize DNA.
2. Inability to Anneal Primers: At such a high temperature, not only would the
polymerase be affected, but also there would be insufficient conditions for primer
annealing to occur on the template DNA. Primers need a lower temperature to bind
effectively.
3. Overall Reaction Failure: The combination of these factors would likely result in a
complete failure of the PCR process, preventing any amplification of the target DNA
sequence.
The lab manual shows the PCR steps include 72°C for elongation, so going way higher than
that would definitely screw things up.
QUESTION 4: Why does a PCR reaction require a primer? Would you expect this primer to
be composed of DNA or RNA? Explain your reasoning. [3 marks]
A PCR reaction requires primers because they provide a starting point for DNA synthesis.
Primers are short sequences of nucleotides that bind specifically to complementary regions on
the target DNA template. Without primers, DNA polymerase would have no way to initiate
synthesis since it can only add nucleotides to an existing strand.
Primers are composed of DNA rather than RNA for several reasons:
1. Stability: DNA is more stable than RNA at higher temperatures used in PCR, which
helps maintain primer integrity during thermal cycling.
2. Specificity: DNA primers can be designed with specific sequences that match target
regions in the template DNA, increasing specificity in amplification.
3. Compatibility with Polymerase: The enzymes used in PCR are designed to work with
DNA substrates; therefore, using RNA primers would not be effective in this context.
The lab manual shows we're using DNA primers for both the blue and brown eye color
alleles, so that backs this up.
QUESTION 5: Should PCR primers be complementary to each other? Explain the reasoning
for your answer. [2 marks]
PCR primers should not be complementary to each other because if they were, they would
bind together instead of binding to their respective target sequences on the template DNA.
This could result in primer dimer formation, which would consume primers and prevent them
from binding effectively to their intended targets. The design of primers aims for them to
anneal specifically to opposite strands of the target region, facilitating successful
amplification without interference from each other.
QUESTION 6: Based on your knowledge of DNA replication, indicate the polarity of each
primer. [2 marks, 0.5 mark each]
Regarding primer polarity:
● Forward Primer: 5' to 3'
● Reverse Primer: 5' to 3'
● Template DNA (sense strand): 3' to 5'
● Template DNA (antisense strand): 3' to 5'
