Rohan Nath

19758451`

01

Mon 2pm

PCR assignment

500 words

30th aug

30th aug

Campbelltown/online

Rohan Nath
The polymerase chain reaction (PCR) is a technique used in scientific labs to amplify DNA sequences.
The method selects the genomic area to be amplified by using short DNA sequences known as
primers. To help a DNA replication enzyme reproduce the correct sequence, the temperature of the
sample is increased and lowered repeatedly. The technique may create a billion replicas of the
necessary sequence in only a few hours 1. Each PCR experiment necessitates the use of template
DNA, primers, nucleotides, and DNA polymerase. The DNA polymerase enzyme, on the other hand,
is the primary enzyme that joins individual nucleotides to form the PCR product 2. Adenine, thymine,
cytosine, and guanine (A, T, C, G) are the four bases found in DNA and serve as the building blocks
for the DNA polymerase to use in generating the PCR result. A primer is a short sequence of DNA
nucleotides, usually 18 to 24 base pairs long, having a complementary sequence to the target DNA to
be detected and amplified. Nonetheless, the primers have been evaluated to recognise a sequence
located upstream complementary strand (3′–5′) of the same fragment DNA complementarily; the
other to detect a sequence located upstream complementary strand (3′–5′) of the same fragment
DNA complementarily. The reaction primers, on the other hand, specify the precise DNA product to
be amplified and may also be used in a variety of experimental procedures to provide a starting
point for the DNA polymerase to build on 3.

PCR is a common diagnostic tool for detecting pathogens, such as viruses that cause diseases such as
Human Papillomaviruses. The combination of PGMY11/09 (PGMY) consensus primers (figure 1)
provide better sensitivity in detecting and typing human papillomavirus (HPV) DNA by PCR. The
polymorphic L1 viral genomic region is amplified by PCR utilising multiplex primers in HPV testing.
HPV PCR amplicons, on the other hand, have been proven to successfully discriminate between HPV
types4.The DNA from patient samples must be extracted and amplified with the HPV plasmid using a
Qiagen Multiplex PCR kit, which includes HotStarTaq DNA Polymerase and a one-of-a-kind PCR
buffer including the novel synthetic Factor MP5 (Figure 2). This additive stabilises primarily bound
primers and enables for a realistic extension of all primers in the process without the need for
optimization when paired with suitable salt concentrations 6. For the simultaneous synthesis of a
human 3-globin product of 260 bp that served as an endogenous control, the human beta-globin
gene PCR primers were 5′ GAAGAGCCAAGGACAGGTAC-3′ (GH20; forwards) and 5′
CAACTTCATCCACGTTCACC-3′ (PC04; reverse)5.

Each reaction, on the other hand, was subjected to 40 amplification cycles in a DNA Thermal Cycler,
with thermocycler-step values of 95 °C for 1 minute, 55 °C for 1 minute, and 72 °C for 2 minutes. The
last 72 °C elongation cycle was extended for an additional 5 minutes. Electrophoresis on 7%
polyacrylamide gels separated the amplification products, which were then seen under UV light
following ethidium bromide staining7. The result was declared positive for DNA-HPV when one of the
MY09/MY11 oligonucleotide primers tested detected viral DNA 8.
Figure 1: Shows how to make human papillomavirus (HPV) L1 amplicon pools using primer sets. A
464-nucleotide product is generated by combining PGMY11/09 PGMY consensus primers (nt). This
assists in the detection of HPV7

Figure 2: Factor MP improves

the local concentration of
primers around the template,
allowing HotStarTaq Plus DNA
Polymerase to efficiently
lengthen primers, resulting in
more effective primer/probe
hybridization to the template5.
 Refrence:

1. Garibyan L, Avashia N. Polymerase Chain Reaction. Journal of Investigative Dermatology.

2013;133(3):1-4.
2. Templeton N. The Polymerase Chain Reaction History Methods, and Applications.
Diagnostic Molecular Pathology. 1992;1(1):58-72.
3. Venceslau E, Bezerra M, Lopes A, Souza É, Onofre A, Melo C et al. HPV detection using
primers MY09/MY11 and GP5+/GP6+ in patients with cytologic and/or colposcopic
changes. Jornal Brasileiro de Patologia e Medicina Laboratorial. 2014;50(4).
4. Resnick R, Cornelissen M, Wright D, Eichinger G, Fox H, Schegget J et al. Detection and
Typing of Human Papillomavirus in Archival Cervical Cancer Specimens by DNA
Amplification With Consensus Primers. JNCI Journal of the National Cancer Institute.
1990;82(18):1477-1484.
5. IAGEN Multiplex PCR Kit [Internet]. Qiagen.com. 2021 [cited 27 August 2021]. Available
from: https://www.qiagen.com/au/shop/pcr/qiagen-multiplex-pcr-kit/#productdetails
6. Ritari J, Hultman J, Fingerroos R, Tarkkanen J, Pullat J, Paulin L et al. Detection of Human
Papillomaviruses by Polymerase Chain Reaction and Ligation Reaction on Universal
Microarray. PLoS ONE. 2012;7(3):e34211.
7. Yin L, Yao J, Chang K, Gardner B, Yu F, Giuliano A et al. HPV Population Profiling in Healthy
Men by Next-Generation Deep Sequencing Coupled with HPV-QUEST. Viruses.
2016;8(2):28.
8. Kadri K. Polymerase Chain Reaction (PCR): Principle and Applications. Synthetic Biology -
New Interdisciplinary Science. 2020;.