PCR & GE Reviewer

PCR & Gel Electrophoresis Reviewer

POLYMERASE CHAIN REACTION (PCR) What is PCR?

Invented in 1983 by Dr. Kary Mullis. It's an in-vitro technique for amplifying a region of DNA whose sequence is known. It works by using DNA polymerase to synthesize new strands complementary to a template strand — but it needs a primer to start.

PCR Components (memorize all 7)

  1. Template DNA — target DNA to be amplified; up to 3Kb; 0.1–1 µg in a 50 µL reaction

  2. Forward primer (upstream) — complementary to 3' end of the antisense strand (3'→5')

  3. Reverse primer (downstream) — complementary to 3' end of the sense strand (5'→3')

  4. Taq DNA Polymerase — heat-stable enzyme that performs extension; ~1.25 U per 50 µL reaction

  5. Buffer — stabilizes all components; 500 mM KCl + 100 mM Tris-HCl (pH 8.3)

  6. Magnesium Chloride (MgCl₂) — essential cofactor of DNA polymerase; 0.5–3.5 µM; too little = no enzyme activity; too much = non-specific amplification

  7. dNTPs — dATP, dGTP, dCTP, dTTP in equimolar amounts; stored at 10 mM pH 7.0; add 20–200 µM in assay

When all components are mixed together, the mixture is called a PCR mix / mastermix / cocktail. If even ONE component is missing, NO amplification will occur.

Primer Details

  • Length: 15–30 nucleotides

  • GC content: 40–60%

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

  • When "1 primer" is mentioned, it always means 1 pair = forward + reverse = 2 total

  • Both strands of dsDNA must be targeted, so both primers must always be present

Polymerase Variants

Polymerase

Source Organism

Taq

Thermus aquaticus

Pfu

Pyrococcus furiosus

KOD

Thermococcus kodakarensis KOD1

3 Types of Amplification Techniques

  • Target amplification

  • Signal amplification

  • Probe amplification

PCR CYCLE STEPS Table 6.2 — MEMORIZE THIS

Step

Temperature (°C)

Time (sec)

Denaturation

90–96

20–60

Annealing

50–70

20–90

Extension

68–75

10–60

Values may differ per publication but should fall within these ranges.

Step 1: Denaturation (90–96°C)

  • Temperature rises to melt hydrogen bonds between the two DNA strands

  • Converts dsDNA → ssDNA

  • This is reflected as the rising line on the PCR cycle graph

Step 2: Annealing (50–70°C; most critical step)

  • Temperature gradually decreases to ~55–65°C

  • Primers bind to their complementary sequences on the single-stranded template

  • This is the most critical stage — each primer has a specific annealing temperature

  • Denaturation and extension temperatures are usually the same across primers, but annealing temperature varies per primer

Step 3: Extension (68–75°C)

  • DNA polymerase and dNTPs extend the primer

  • Maximum temperature is 75°C — exceeding this causes denaturation instead

  • Temperature slightly increases from annealing step

One complete cycle = Denaturation + Annealing + Extension
Standard number of cycles: 30–35 cycles
After 35 cycles → billions of copies = exponential amplification

CONVENTIONAL PCR vs. REAL-TIME PCR

Feature

Conventional PCR

Real-Time PCR (qPCR)

How it works

Denature → Anneal → Extend; repeated cycles

Uses fluorescent dyes/probes to monitor amplification in real-time

Quantification

No — requires post-PCR analysis (gel electrophoresis)

Yes — based on comparison with standard curves

Sensitivity

Lower

Higher

Contamination risk

Higher (multiple handling steps)

Lower (closed-tube format)

Advantage

Simple, widely used; versatile (cloning, sequencing, genotyping)

Provides quantitative data; wider range of applications

Disadvantage

Cannot detect low copy number targets

High initial setup costs; vulnerable to PCR inhibitors

CT Value (Cycle Threshold) — Real-Time PCR

  • The CT value = the specific cycle at which fluorescence signal starts to rise (amplification begins)

  • Rule of thumb:

    • Lower CT = Higher initial DNA concentration (amplifies early, cycles 15–20)

    • Higher CT = Lower initial DNA concentration (amplifies late, >20 cycles)

  • Early cycles: 15–20

  • Late cycles: >20 (up to max cycle number)

  • Samples amplifying beyond the set cycle number = considered negative or contaminated or non-specific

OTHER TYPES OF PCR

PCR Type

How It Works

Advantages

Disadvantages

RT-PCR

Reverse transcriptase converts RNA → cDNA → conventional PCR

Amplifies/analyzes RNA; useful for RNA viruses & gene expression

Affected by RNA integrity; risk of non-specific amplification

Multiplex PCR

Multiple primer sets for different targets in one reaction

Saves time; detects multiple pathogens simultaneously

Complex primer design; harder to optimize

GEL ELECTROPHORESIS

Principle

Charged molecules (DNA, proteins) migrate in response to an electrical field. Performed after PCR to visualize amplified DNA — especially necessary after conventional PCR since you cannot see amplification in real time.

Factors Affecting Migration Rate

1. Strength of Field (Voltage)

  • Standard: 100 V

  • High voltage → faster migration but generates heat → distorts gel and reduces resolution (blurry bands)

  • If resolution is poor, try lowering to 80 or 50 V with longer run time

2. Ionic Strength & Buffer Composition

  • Ions in the buffer conduct electricity evenly

  • Too little ions → no electrical conduction → no migration

  • Too much → excess heat → gel distortion

  • Standard buffer concentrations:

    • TBE (Tris-Borate-EDTA): stock = 10x

    • TAE (Tris-Acetate-EDTA): stock = 50x

    • In SPC lab: 0.25x concentration is used

3. Viscosity

  • Buffer should NOT be viscous — viscosity = high concentration = slowed migration

4. Temperature

  • Should not be too hot — excess heat distorts gel and reduces resolution sharpness

5. Size of Molecule

  • Smaller = lighter → faster migration → farther from well

  • Larger = heavier → slower migration → closer to well

  • Separation in gel electrophoresis is based on SIZE, not charge

6. Shape

  • DNA is normally linear

  • Compact/globular → travels faster

  • Elongated/irregular → slower migration

7. Net Charge

  • DNA is negatively charged (anion)

  • Migrates from cathode (–) → anode (+)

  • Migration is based on charge; separation is based on size

8. Agarose Concentration

  • Higher agarose concentration → smaller pores → better for small molecules

  • Lower agarose concentration → larger pores → better for large/complex DNA

Agarose Concentration Table

Agarose (%)

DNA Size Range

0.5%

700 bp – 25 kb

0.8%

500 bp – 15 kb

1.0%

250 bp – 12 kb ← standard

1.2%

150 bp – 6 kb

1.5%

80 bp – 4 kb

Standard is 1% as a "safety net" — works for both small and large molecules.

Types of Electrophoresis

Feature

Agarose Gel (AGE)

Polyacrylamide Gel (PAGE)

Orientation

Horizontal

Vertical

Pore size

Bigger

Smaller

Best for

Large molecules

Small molecules

Resolves small size differences?

No

Yes

Molecules run

Mostly DNA

DNA or proteins

Types: AGE, PAGE (including SDS-PAGE), Starch gel electrophoresis

DNA Staining

Ethidium Bromide (EtBr)

  • Intercalates into DNA's planar structure

  • UV absorbed by DNA at 160 nm is transmitted to the dye

  • Emits red-orange fluorescence at 590 nm — visible to the naked eye under UV

Alternative: Gel Red

Reading Gel Results

The Ladder

  • Also called a DNA marker

  • Placed in the first well as a size reference for all other bands

  • Appears as a ladder-like pattern (e.g., 5000 bp, 1500 bp, 500 bp)

Interpreting Band Quality

  • Intact, sharp bands → good DNA quality

  • Smeared or smudged bands → degraded DNA, poor quality, or contamination

Interpreting Band Position

  • Compare sample band position to the ladder

  • If expected amplicon size matches the band position → successful amplification

  • If band appears at wrong position → unsuccessful; likely non-specific primer binding

Interpreting Specificity

  • Only ONE band at the correct size → most specific result (ideal)

  • Multiple bands → contamination or non-specific primer

Controls in PCR/Gel Electrophoresis

Negative Control

  • Contains ALL PCR components EXCEPT the DNA template

  • Must ALWAYS be included — it cannot be skipped

  • Expected result: NO band

  • Purpose: to detect contamination

  • Must be processed at the same time as the actual sample

  • If a band appears in the negative control → PCR is INVALIDATED → repeat the entire run

Positive Control

  • Optional (expensive but useful)

  • Confirms primer is working correctly

  • Can be skipped if budget is limited since the ladder already provides size reference for specificity

Key Reminders

  • Gel electrophoresis is performed after conventional PCR because you cannot visualize amplification without it

  • Real-time PCR does not require gel electrophoresis since it monitors amplification in real time

  • If the negative control shows a band (even faint), the sample result is invalid

  • The primer determines the amplicon size — this is how you know if the correct gene was amplified