PCR (Polymerase Chain Reaction)
test
I. What is PCR?
PCR (Polymerase Chain Reaction) is a molecular biology technique used to amplify specific
DNA sequences in vitro. It allows the creation of millions of copies of a DNA fragment from a
very small starting amount.
Invented by: Kary Mullis in 1983
Key enzyme: Taq DNA polymerase (from Thermus aquaticus)
II. Principle of PCR
PCR relies on repeated cycles of:
1. Denaturation – DNA strands separate
2. Annealing – Primers bind to target DNA
3. Extension – DNA polymerase synthesizes new DNA strands
Each cycle doubles the amount of DNA, resulting in exponential amplification.
III. Components of a PCR Reaction
Component Function
Template DNA The target DNA to be
amplified
Primers Short synthetic DNA
sequences (forward &
reverse) that flank the
target region
DNA polymerase Enzyme that synthesizes
new DNA (e.g., Taq)
dNTPs Building blocks (A, T, G, C)
Buffer Maintains optimal pH and
ion concentrations
MgCl₂ Cofactor for polymerase
activity
IV. PCR Cycle Steps
1. Denaturation (~94–98°C for 15–30 sec)
Separates the double-stranded DNA
2. Annealing (~50–65°C for 15–60 sec)
Primers bind to complementary sequences on DNA
3. Extension (~72°C for 30–60 sec)
DNA polymerase adds nucleotides to synthesize the new strand
🔁 These steps are repeated for 25–40 cycles
V. Types of PCR
Type Description Example Use
Conventional PCR Basic Pathogen
amplification, detection
results analyzed
by gel
electrophoresis
RT-PCR Reverse Viral RNA
Transcription PCR detection (e.g.,
– starts with RNA HIV)
and converts it to
cDNA
qPCR (Real-Time) Quantifies DNA Viral load, gene
during expression
amplification
using
fluorescence
Multiplex PCR Multiple primers Respiratory
used to amplify pathogen panels
several targets
simultaneously
Nested PCR Two sets of Detecting low-
primers to copy targets
increase
specificity
Digital PCR Partitions sample Precision
into many micro- diagnostics
reactions for
absolute
quantification
VI. Applications of PCR
1. Medical Diagnostics
Infectious diseases: COVID-19, HIV, Hepatitis B/C, TB
Genetic diseases: Cystic fibrosis, sickle cell anemia
Cancer: Detection of mutations (e.g., BCR-ABL in leukemia)
2. Forensic Science
DNA fingerprinting for criminal investigations and paternity testing
3. Research
Gene expression analysis
Cloning and sequencing
4. Agriculture & Food Safety
GMO detection
Pathogen screening in food
VII. Interpretation of PCR Results
For Conventional PCR:
Products are run on agarose gel electrophoresis
DNA bands are visualized using ethidium bromide or SYBR Green under UV light
Observation Interpretation
Band at expected size Positive result
No band Negative result or failed
reaction
Non-specific bands Poor primer design or
contamination
For qPCR:
Results shown as Ct (Cycle threshold) values:
Lower Ct = Higher initial DNA concentration
Higher Ct = Lower starting material
VIII. Advantages of PCR
✅ Highly sensitive (detects small amounts of DNA)
✅ Highly specific (uses sequence-specific primers)
✅ Rapid results (within hours)
✅ Versatile for DNA and RNA targets
IX. Limitations of PCR
❌ Prone to contamination → false positives
❌ Requires precise temperature control
❌ Not suitable for quantification unless using qPCR
❌ Amplifies even dead microorganisms, so may not reflect active infection
X. Common PCR Troubleshooting
Problem Possible Cause
No amplification Missing reagent, degraded
template, primer mismatch
Multiple/non-specific bands Poor primer design, too low
annealing temp
Weak bands Low template
concentration, insufficient
cycles
Smearing on gel Degraded DNA, excessive
template, contamination
XI. Summary Chart: Key PCR Types
PCR Type Template Output Use Case
PCR DNA Amplified General
DNA detection
RT-PCR RNA cDNA → DNA Detect RNA
viruses (e.g.
HIV)
qPCR DNA/cDNA Quantitative Viral load,
Ct gene
expression
Multiplex DNA Multiple Panel testing
PCR targets
