PCR Variations
DEFINITIONS (qPCR-Related Terms)
Ct (Cycle Threshold)
The PCR cycle number at which fluorescence rises above background detection in qPCR.
Lower Ct = higher starting template amount
6_PCR (1)
(Section 10: Real-Time PCR).
Fluorophore
A fluorescent molecule that emits light when excited by a specific wavelength. Used in qPCR probes (e.g., SYBR Green, TaqMan).
Quencher
A molecule positioned near a fluorophore that suppresses fluorescence when in close proximity.
FRET (Fluorescence Resonance Energy Transfer)
Energy transfer between fluorophore and quencher when they are very close together.
When separated (e.g., probe cleavage), fluorescence becomes detectable.
Molecular Beacon
A hairpin-shaped oligonucleotide probe with:
Fluorophore on one end
Quencher on the other
When not bound → hairpin keeps fluorophore and quencher close → no signal.
When bound to target → hairpin opens → fluorescence emitted
Traditional PCR vs Colony PCR
Traditional PCR
Extract DNA from cells
Add template DNA to PCR mix
Run thermal cycling
Analyze by gel electrophoresis
Colony PCR
Pick bacterial colony
Directly add small amount to PCR tube
Initial denaturation lyses cells
Amplify target region
Gel analysis to confirm insert presence
Difference:
Colony PCR skips DNA purification and uses bacterial cells directly.
qPCR (Real-Time PCR)
Basic Steps:
Standard PCR cycling (denature → anneal → extend)
Fluorescence measured every cycle
Amplification curve generated
Ct value calculated
Detection chemistries:
SYBR Green (binds dsDNA)
Probe-based systems
qPCR with 5′ Nuclease Probes (TaqMan)
Mechanism:
Probe binds between primers
Probe has fluorophore (5′) and quencher (3′)
During extension, Taq polymerase cleaves probe
Fluorophore separates from quencher
Fluorescence increases proportionally to product
Advantage: Higher specificity than SYBR Green.
qPCR with Molecular Beacons
Mechanism:
Beacon is hairpin-shaped in solution
When target binds, hairpin opens
Fluorophore separated from quencher
Signal emitted
Unlike TaqMan, beacon is not degraded.
Multiplex PCR
Multiple primer sets in same reaction:
Amplifies several targets simultaneously
Requires careful primer design (similar Tm)
Often uses differently colored fluorophores in qPCR
Used for pathogen panels, genotyping.
TMA (Transcription-Mediated Amplification)
Isothermal amplification (no thermal cycling).
Steps:
Reverse transcriptase converts RNA → cDNA
RNA polymerase produces many RNA copies
Amplification is exponential
Primarily used for RNA detection (e.g., viral diagnostics).
LAMP (Loop-Mediated Isothermal Amplification)
Isothermal (constant ~60–65°C).
Features:
Uses 4–6 primers
Strand-displacing DNA polymerase
Produces large amounts of DNA rapidly
Visual detection possible (turbidity/color change)
Advantages:
No thermocycler required
Fast (30–60 min)
Conventional End-Point PCR vs qPCR vs TMA vs LAMP
Feature | End-Point PCR | qPCR | TMA | LAMP |
|---|---|---|---|---|
Detection | Gel after cycling | Real-time fluorescence | Real-time RNA amplification | Real-time/isothermal |
Quantification | No | Yes (Ct value) | Yes | Semi-quantitative |
Temperature | Thermal cycling | Thermal cycling | Isothermal | Isothermal |
Enzyme | Taq polymerase | Taq polymerase | Reverse transcriptase + RNA polymerase | Strand-displacing polymerase |
Speed | Moderate | Faster | Fast | Very fast |
Equipment | Thermocycler + gel | Real-time PCR machine | Specialized | Heat block only |
Target | DNA | DNA/RNA | Mainly RNA | DNA |
Key Concept Differences
End-Point PCR
Detects final amplicon
Cannot determine starting quantity
qPCR
Detects product accumulation during exponential phase
Provides quantitative data
Ct inversely proportional to template amount
6_PCR (1)
TMA
RNA amplification
No thermal cycling
Used in clinical diagnostics
LAMP
Simple field testing
Rapid detection
High amplification efficiency