PCR Reviewer

PCR Exam Reviewer — MLS 420

PART 1: FUNDAMENTALS

What is PCR?
Invented in 1983 by Dr. Kary Mullis. It's an in-vitro technique that amplifies a specific region of DNA whose sequence is known (or lies between two regions of known sequence). It belongs to Target Amplification — where DNA itself is copied.

The 3 types of amplification techniques are Target Amplification, Signal Amplification (detection signal is increased), and Probe Amplification (probes are amplified).

PART 2: PCR COMPONENTS

1. Template DNA

  • Contains the region to be amplified

  • Up to 3 kb in length

  • Added at 0.1–1 µg in a 50 µL reaction

2. Primers

  • Short DNA sequences (15–30 nucleotides) that define the region to be amplified

  • GC content: 40–60%

  • Concentration: 50 pmol (1 µM final in 50 µL reaction)

  • Complementary to the 3' ends of target DNA

  • Forward primer → complementary to the 3' end of the antisense strand (3'→5')

  • Reverse primer → complementary to the 3' end of the sense strand (5'→3')

3. Buffer

  • Maintains optimal pH and ionic conditions

  • Stabilizes the polymerase, DNA, and nucleotides

  • Contains: 500 mM KCl + 100 mM Tris-HCl (pH 8.3)

4. Magnesium (MgCl₂)

  • Essential cofactor of DNA polymerase

  • Used at 0.5–3.5 µM

  • Too little → no reaction; Too much → non-specific amplification

  • Precautions: completely thaw, vortex before pipetting; be careful if samples contain EDTA (it chelates Mg²⁺)

5. DNA Polymerase (Taq)

  • Heat-stable enzyme responsible for extension

  • ~1.25 U per 50 µL reaction

  • Variants:

    • Taq — from Thermus aquaticus (most common)

    • Pfu — from Pyrococcus furiosus (hyperthermophilic)

    • KOD — recombinant from Thermococcus kodakarensis KOD1

6. dNTPs

  • Building blocks added during Extension

  • dATP, dGTP, dCTP, dTTP in equimolar amounts

  • Stored at 10 mM, pH 7.0; added at 20–200 µM in assay

PART 3: PCR CYCLE STEPS

Step

Temperature

Time

Denaturation

90–96°C

20–60 sec

Annealing

50–70°C

20–90 sec

Extension

68–75°C

10–60 sec

Denaturation — Heat separates (unwinds) the double-stranded DNA into two single strands.

Annealing — Temperature drops; primers bind to their complementary sequences on the template.

Extension — Taq polymerase synthesizes new DNA strands by adding dNTPs to the 3' end of the primers. DNA doubles with each cycle → exponential amplification.

The machine controlling temperature changes is the Thermal Cycler.

PART 4: PCR PROCEDURE (Lab Steps)

Step 1 — Preparation: Thaw all components on ice. Ensures proper concentration and prevents reagent degradation.

Step 2 — Labelling: Properly label all tubes to avoid mix-up and contamination.

Step 3 — PCR Cocktail (Master Mix): Prepare master mix on ice containing all reagents except DNA. Mix by gently pumping micropipette ~20 times.

Master Mix components (per 10 µL): Buffer (1.5), dNTPs (0.3), MgCl (0.3), Forward Primer (0.3), Reverse Primer (0.3), Taq Polymerase (0.2), Molecular Grade Water (7.1).

Step 4 — Add DNA Sample: Transfer 10 µL master mix into each labeled PCR tube, then add DNA. Positive control and negative control get separate 0.2 mL thin-walled tubes. Negative control has NO template DNA.

Step 5 — Run Thermal Cycler:

  • Initial denaturation: 95°C for 3 minutes

  • 35 cycles of: denaturation (95°C/30 sec) → annealing (30 sec) → extension (72°C/1 min per kb)

  • Final extension: 72°C, 1 min per kb

Step 6 — Store Amplicons: Store at 4°C. Analyze via agarose gel electrophoresis.

PART 5: TROUBLESHOOTING

Low yield / No amplification

  • Causes: missed components, pipetting error, suboptimal annealing temp, wrong DNA concentration, reagent problems

  • Fix: repeat PCR, optimize annealing temp (gradient PCR), adjust DNA concentration, increase one reagent at a time (DNA → primer → dNTP → MgCl₂ → Taq), add DMSO or betaine (decreases Tm, inhibits secondary structures)

Amplification in the blank (negative control)

  • Causes: pipetting carry-over, contaminated reagents

  • Fix: repeat PCR, replace pipette tips, avoid reagent contamination

Wrong product size

  • Cause: mispriming (primers annealed at wrong site), non-specific amplification

  • Fix: optimize primer design

Extra bands in electrophoresis

  • Causes: primers annealed at multiple sites, too much MgCl₂/polymerase/primer, non-specific primers

  • Fix: repeat primer design, reduce excess reagents

QUICK MEMORY AIDS

  • PCR invented: 1983, Dr. Kary Mullis

  • Template size: up to 3 kb

  • Primer length: 15–30 nucleotides; GC: 40–60%

  • Taq source: Thermus aquaticus (heat stable!)

  • dNTPs added during: Extension

  • MgCl₂ role: Cofactor — too little = no rxn; too much = non-specific bands

  • Cycle order: Denature → Anneal → Extend (DAE)

  • Result analysis: Agarose gel electrophoresis