1. Define PCR and Thermal Cycler.
• Polymerase Chain Reaction (PCR) is a revolutionary molecular biology technique developed by
Kary Mullis in 1983. It allows for the in vitro amplification of specific DNA sequences by
mimicking the natural DNA replication process in a highly controlled environment. By cycling
through repeated steps of denaturation, annealing, and extension, PCR can exponentially produce
millions to billions of copies of a particular DNA target, even from minuscule amounts of starting
material. This technique is fundamental in medical diagnostics, forensic science, genetic
engineering, and research.
• A Thermal Cycler, also known as a PCR machine, is a sophisticated instrument that automates the
precise temperature changes required during PCR. It rapidly cycles through the denaturation,
annealing, and extension phases, ensuring accuracy and reproducibility. Advanced thermal cyclers
often include heated lids to prevent condensation and may be equipped with fluorescence detection
systems for real-time PCR applications.
2. What is the purpose of PCR?
The principal aim of PCR is to amplify a specific DNA region, making it detectable and analyzable for a
variety of laboratory applications. Its significance in the clinical laboratory cannot be overstated—it allows
for:
• Early and accurate diagnosis of infectious diseases such as HIV, COVID-19, and tuberculosis;
• Genetic screening for inherited disorders;
• Forensic identification through DNA fingerprinting;
• Research applications, such as gene expression studies and molecular cloning;
• Monitoring therapy effectiveness, especially in oncology and virology.
3. Explain and discuss the differences between conventional PCR and real-time PCR/qPCR.
Conventional PCR and Real-Time PCR (qPCR) differ primarily in how and when the amplified DNA is
detected:
• Conventional PCR involves endpoint detection. After all amplification cycles are complete, the
amplified DNA (amplicons) is visualized using gel electrophoresis, often with ethidium bromide
staining under UV light. It provides qualitative results—i.e., the presence or absence of a DNA
sequence.
• qPCR (Quantitative or Real-Time PCR) allows for the real-time monitoring of amplification. It
utilizes fluorescent dyes (e.g., SYBR Green) or sequence-specific probes (e.g., TaqMan) to measure
DNA accumulation at each cycle. This provides quantitative data on DNA concentration, making
qPCR an invaluable tool in clinical diagnostics and research.
4. Discuss the 3 steps of PCR and their relation to thermal cycler.
PCR operates in cycles, each consisting of three critical temperature-dependent steps, all regulated by
the thermal cycler:
1. Denaturation (94–98°C)
oDouble-stranded DNA melts into single strands by breaking hydrogen bonds.
oSets the stage for primers to bind to their targets.
2. Annealing (50–65°C)
o Short primers anneal (bind) to complementary sequences on the single-stranded DNA.
o The exact temperature depends on primer design and GC content.
3. Extension (72°C)
o Taq DNA polymerase, a heat-stable enzyme derived from Thermus aquaticus, synthesizes
new DNA by adding dNTPs to the primers.
This cycle is repeated typically 25–35 times, exponentially increasing the DNA copies. The thermal cycler
ensures precise timing and temperature control for each phase, making PCR efficient, reproducible, and
automated.
5. Discuss the application of conventional PCR and qPCR.
Both conventional and real-time PCR are powerful tools in the arsenal of a modern medical technologist:
Conventional PCR Applications:
• Infectious disease diagnostics (e.g., Mycobacterium tuberculosis)
• Mutation detection (e.g., cystic fibrosis gene mutations)
• Molecular cloning in research
• Forensic DNA profiling
qPCR Applications:
• Quantification of viral load (e.g., HIV, SARS-CoV-2)
• Gene expression analysis (using reverse-transcribed mRNA)
• Cancer biomarker detection (e.g., HER2 amplification)
• Pharmacogenomic studies for personalized medicine
qPCR has the added advantage of being more sensitive, faster, and suitable for quantitative clinical
reporting, especially in monitoring disease progression and treatment response.
6. What type of PCR will you use when your sample is RNA? Differentiate it from the conventional
PCR.
When working with RNA samples, the appropriate technique is Reverse Transcription PCR (RT-PCR).
• In RT-PCR, RNA is first converted into complementary DNA (cDNA) using the enzyme reverse
transcriptase. The cDNA then serves as the template for conventional PCR amplification.
• This is essential for detecting and analyzing RNA viruses (like SARS-CoV-2) or measuring gene
expression via mRNA.
Comparison with Conventional PCR:
RT-PCR is often coupled with qPCR for RT-qPCR, combining both reverse transcription and real-time
monitoring—a gold standard for viral diagnostics.
Feature Conventional PCR RT-PCR
Starting material DNA RNA
Additional step None Reverse transcription to cDNA
Use case DNA detection RNA detection, gene expression
Enzyme used Taq polymerase Reverse transcriptase + Taq
References:
1. Thermo Fisher Scientific. (n.d.). PCR Basics: What is PCR?. Retrieved from
https://www.thermofisher.com
2. National Human Genome Research Institute. (n.d.). Polymerase Chain Reaction Fact Sheet.
https://www.genome.gov
3. Enzo Life Sciences. (n.d.). Differences Between PCR, RT-PCR, qPCR and RT-qPCR.
https://www.enzo.com
4. LabCE Continuing Education. (n.d.). PCR Techniques in the Clinical Laboratory.
https://www.labce.com
5. YouTube Videos:
o PCR animation: https://youtu.be/rpL5vEbOmgc
o Thermal cycler explanation: https://youtu.be/a5jmdh9AnS4
o Conventional PCR steps: https://youtu.be/db0HzFTJtCs
o Real-Time PCR overview: https://youtu.be/wBrNbbAlAFo
o RT-PCR vs. qPCR: https://youtu.be/8GOKaz8MRyM
