95 February 26, 2019

MARASIGAN, Jhoellah D. March 5, 2019

Group # 12

EXPERIMENT NO. 16
POLYMERASE CHAIN REACTION
Answers to Guide Questions
1. What are the three major steps in a thermal cycling profile of simple PCR? Describe what
happens in each step. (6 points)
a. Denaturing - Double-stranded DNA is heated to separate it into individual strands by
raising the temperature of the PCR solution to just below boiling so all the hydrogen
bonds holding the complementary strands of DNA together are broken. 1, 2, 3
b. Annealing - The temperature is lowered to enable the DNA primers to attach to the
template DNA or the reassociation of primers to form a double helix when the
temperature is lowered below Tm. 1, 2, 3
c. Extending - The solution is then heated to 72°C, the optimal temperature for Taq
DNA polymerase, then it elongates both primers in the direction of the target
sequence because DNA synthesis is in the 5’-to- 3’ direction. 1, 2, 3

2. A. what is the purpose of the long incubation time at the initial denaturation part of thermal
cycling profile? (2 points)
Mammalian DNA may necessitate longer incubation periods compared to plasmids and PCR
products, based on their DNA complexity and size. Complete denaturation of the DNA aids
guarantees in effective amplification of the target sequence during the first amplification cycle.
4

B. what is the purpose of the final extension step (which is usually 6 to 10 minutes) in the
thermal cycling profile? (2 points)
Increasing the final extension time improves full-length replication and according to
thermofisher, final extension also provides yield of a 0.7-kb, GC-rich PCR fragment from
human gDNA in these experiments. 4
 3. A. In a PCR master mix, what is the function of each of the following components: primer,
dNTP Taq polymerase, DNA template. (4 points)
i. Primer – This contains the sequences complementary to the target area to enable
selective and repeated amplification. 1, 4
ii. dNTP - These are the building-blocks from which the DNA polymerase creates a new
DNA strand. It is vital that equal amounts of each nucleotide (dATP, dTTP, dCTP
and dGTP) are added to the master mix to avoid mismatches of bases. 1, 4
iii. Taq polymerase – This permits elongation of the DNA by attaching to the primer and
then adding DNA bases to the single strand one-by-one in the 5’ to 3’ direction. 1, 4
iv. DNA template - which will be amplified by the PCR reaction 1, 4
B. MgCl2 is a component of the PCR buffer, what is its function? (2 points)
MgCl2 serves as a cofactor for the enzyme. Magnesium ions facilitate establishment of the
complex between the primers and DNA templates by stabilizing negative charges on
phosphate backbones. 5, 6

4. What are the characteristics of a good PCR product when examined by gel electrophoresis? (3
points)
a. Absence of no or poorly visible bands 7
b. No smeared or diffuse (fuzzy) bands 7
c. No poorly separated bands 7

5. Describe 3 important applications of PCR in laboratory medicine.

a. PCR can be used in paternity testing or DNA profiling. 8
b. PCR can be used in detecting gene mutation. 8
c. PCR can be used in forensic analysis. 8

References:
1. Murray, Robert et al. Harpers Illustrated Biochemistry 29Th Edition. Mcgraw-Hill Publishing, 2012.
2. "What Is PCR (Polymerase Chain Reaction)?". Yourgenome.Org, 2019,
https://www.yourgenome.org/facts/what-is-pcr-polymerase-chain-reaction. Accessed 4 Mar 2019.
3. Berg, Jeremy Mark et al. Biochemistry. W.H. Freeman, 2002.
4. "PCR Cycling Parameters—Six Key Considerations For Success | Thermo Fisher Scientific - UK".
Thermofisher.Com, 2019, https://www.thermofisher.com/ph/en/home/life-science/cloning/cloning-
learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-
cycling-considerations.html. Accessed 4 Mar 2019.
5. "5 Key PCR Components And Their Functions - Biology". Biology.Reachingfordreams.Com, 2019,
http://biology.reachingfordreams.com/biology/molecular-genetics/methods-in-molecular-
genetics/89-pcr-components-and-their-functions.
6. Lorenz, Todd C. "Polymerase Chain Reaction: Basic Protocol Plus Troubleshooting And Optimization
Strategies". Journal Of Visualized Experiments, no. 63, 2012. Myjove Corporation, doi:10.3791/3998.
Accessed 4 Mar 2019.
7. "Nucleic Acid Electrophoresis Troubleshooting Guide | Thermo Fisher Scientific - UK".
Thermofisher.Com, 2019, https://www.thermofisher.com/ph/en/home/life-science/cloning/cloning-
learning-center/invitrogen-school-of-molecular-biology/na-electrophoresis-education/na-
electrophoresis-troubleshooting.html.
8. "PCR (Polymerase Chain Reaction) | LSR | Bio-Rad". Bio-Rad.Com, 2019, http://www.bio-
rad.com/en-ph/applications-technologies/pcr-polymerase-chain-reaction?ID=LUSNYI15. Accessed
4 Mar 2019.