PCR & GE Reviewer (HY)

QUICK FACTS TO MEMORIZE FIRST

PCR invented: 1983, Dr. Kary Mullis
PCR = in-vitro DNA amplification
Standard cycles: 30–35
After 35 cycles = billions of copies (2³⁵ = 68 billion)
Missing even ONE component = NO amplification

PCR COMPONENTS — The 7 Essentials

Component	Key Detail
Template DNA	Up to 3Kb; 0.1–1 µg per 50 µL
Forward primer	Binds antisense strand (3'→5'); upstream
Reverse primer	Binds sense strand (5'→3'); downstream
Taq DNA Polymerase	Heat-stable; 1.25 U per 50 µL
Buffer	500 mM KCl + 100 mM Tris-HCl pH 8.3
MgCl₂	0.5–3.5 µM; too little = no activity; too much = non-specific
dNTPs	dATP, dGTP, dCTP, dTTP; equimolar; 20–200 µM in assay

PRIMERS — High Yield Details

Length: 15–30 nucleotides
GC content: 40–60%
Concentration: 50 pmol (1 µM final)
"1 primer" = always 2 total (forward + reverse)
Both primers must ALWAYS be designed together
Primer determines the amplicon size of target DNA

Forward vs. Reverse

Forward (upstream) → binds antisense strand → goes right
Reverse (downstream) → binds sense strand → goes left

TAQ POLYMERASE VARIANTS

Variant	Source
Taq	Thermus aquaticus ← most important
Pfu	Pyrococcus furiosus
KOD	Thermococcus kodakarensis KOD1

THE 3 PCR STEPS — MEMORIZE THE TABLE

Step	Temp (°C)	Time (sec)	What Happens
Denaturation	90–96	20–60	Hydrogen bonds melt → dsDNA becomes ssDNA
Annealing	50–70	20–90	Primers bind to template; most critical step
Extension	68–75	10–60	Taq extends primer; max temp = 75°C

Values differ per publication but stay within these ranges.

Key Notes Per Step

Denaturation

Goal: separate dsDNA into ssDNA by melting hydrogen bonds
Temperature rises = line rises on graph

Annealing

Most critical stage of PCR
Temperature gradually decreases to ~55–65°C
Each primer has a UNIQUE annealing temperature
Denaturation and extension temps are usually the same across primers — annealing differs

Extension

Taq polymerase + dNTPs do the work
MAX = 75°C — exceeding this causes denaturation instead
Temperature slightly increases from annealing

CONVENTIONAL vs. REAL-TIME PCR

	Conventional PCR	Real-Time PCR (qPCR)
Visualization	Needs gel electrophoresis AFTER	Fluorescent dye — seen DURING
Quantification	No	Yes
Sensitivity	Lower	Higher
Contamination risk	Higher	Lower (closed tube)
Key feature	Versatile; widely used	CT value monitoring

CT VALUE — Real-Time PCR

CT (Cycle Threshold) = the cycle at which fluorescence signal starts to rise

The Golden Rule

CT Value	DNA Concentration
LOW CT	HIGH initial DNA (amplifies early)
HIGH CT	LOW initial DNA (amplifies late)

Early cycles = 15–20
Late cycles = >20
Samples amplifying BEYOND the set cycle limit = negative or contaminated or non-specific
Lowest CT value = highest DNA concentration = amplifies earliest
Highest CT value = lowest DNA concentration = amplifies latest

OTHER PCR TYPES

Type	Key Mechanism	Advantage	Disadvantage
RT-PCR	RNA → cDNA via reverse transcriptase → conventional PCR	Detects RNA viruses; gene expression studies	Affected by RNA integrity
Multiplex PCR	Multiple primer sets in ONE reaction	Detects multiple targets simultaneously	Complex primer design; harder to optimize

GEL ELECTROPHORESIS

Core Principle

Charged molecules migrate through gel in response to an electrical field. Done AFTER conventional PCR to visualize DNA (you cannot see PCR products otherwise).

DNA Migration Direction

DNA = negatively charged (anion)
Migrates: cathode (−) → anode (+)
Separation = based on SIZE
Migration = based on CHARGE

Size vs. Migration — Critical Concept

Fragment	Weight	Migration Speed	Position on Gel
Small/short	Light	Faster	Farther from well
Large/long	Heavy	Slower	Closer to well

FACTORS AFFECTING MIGRATION RATE

1. Voltage

Standard: 100 V
High voltage → faster migration BUT generates heat → distorts gel → blurry bands
Fix: lower to 80 or 50 V with longer run time

2. Buffer (Ionic Strength)

Too little ions → no electrical conduction → no migration
Too much → excess heat → gel distortion
Standard concentrations:

Buffer	Stock	Working
TAE (Tris-Acetate-EDTA)	50x	0.5–1.0x
TBE (Tris-Borate-EDTA)	10x	0.25x
SPC lab TAE	—	0.25x

3. Viscosity

Viscous buffer = highly concentrated = slows migration

4. Temperature

Too hot = distorts gel = poor resolution

5. Size

Smaller = lighter = faster = farther from well
Larger = heavier = slower = closer to well

6. Shape

Compact/globular → faster
Elongated/irregular → slower

7. Agarose Concentration

Higher concentration = smaller pores = better for small molecules
Lower 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 = 1% = works for both small and large DNA = "safety net"

AGAROSE vs. POLYACRYLAMIDE GEL

Feature	Agarose	Polyacrylamide
Orientation	Horizontal	Vertical
Pore size	Bigger	Smaller
Best for	Large molecules	Small molecules
Resolves small differences?	No	Yes
Molecules	Mostly DNA	DNA or proteins

Types: AGE, PAGE, SDS-PAGE, Starch gel electrophoresis

DNA STAINING

Ethidium Bromide (EtBr)

Intercalates into DNA's planar structure
UV absorbed at 160 nm → transmitted to dye
Emits red-orange fluorescence at 590 nm → visible to naked eye
Alternative: Gel Red

READING GEL RESULTS

The Ladder (DNA Marker)

Always placed in the first well
Serves as size reference for all samples
Looks like a ladder (hence the name)
Example rungs: 5000 bp, 1500 bp, 500 bp

Band Quality

Appearance	Interpretation
Sharp, intact bands	Good DNA quality
Smeared/smudged bands	Degraded DNA or contamination present

Band Position Interpretation

Scenario	Interpretation
Band at expected amplicon size	Successful amplification
Band at wrong size	Unsuccessful; non-specific primer
Only ONE band at correct size	Most specific result (ideal)
Multiple bands	Contamination or non-specific primer

CONTROLS — VERY HIGH YIELD

Negative Control

Contains ALL PCR components EXCEPT DNA template
Must ALWAYS be present — never skip this
Processed at the same time as actual sample
Expected result: NO band
Purpose: detect contamination
If a band appears (even faint) → PCR is INVALIDATED → must repeat

Positive Control

Optional (expensive)
Can be skipped if budget is limited
Ladder + known amplicon size is already specific enough without it

HIGH YIELD ONE-LINERS

PCR mix = mastermix = cocktail = all 7 components combined
"1 primer" = 2 primers total (forward + reverse)
Annealing = most critical step of PCR
75°C = maximum extension temperature
30–35 cycles = standard; produces billions of copies
Low CT = high DNA; High CT = low DNA
DNA separates by SIZE but migrates by CHARGE
DNA is an anion → moves toward anode
Small DNA = fast = farther from well
Large DNA = slow = closer to well
1% agarose = standard concentration
Negative control bands = PCR invalidated
Smeared bands = degraded or contaminated DNA
Blurry/distorted gel = wrong voltage or too much heat