| CUE COLUMN | NOTE-TAKING COLUMN |
|---|---|
| Inventor and Nobel Prize | Kary Mullis — 1983 invention, 1993 Nobel Prize in Chemistry. |
| What does PCR amplify? | Any specific DNA sequence flanked by known primer sequences, amplified in vitro from even a single template molecule. |
| Denaturation | 94-98°C, 15-30 sec. Heat breaks hydrogen bonds between complementary base pairs → dsDNA → ssDNA. |
| Annealing | 50-65°C, 20-40 sec. Forward and reverse primers hybridize to their complementary sequences on the single-stranded template. Primer Tm determines optimal temperature. |
| Extension | 72°C, variable (~1 min per 1 kb). Taq polymerase synthesizes new DNA strands 5'→3', incorporating dNTPs. |
| Taq polymerase | From Thermus aquaticus (hot spring bacterium). Thermostable — survives 94-98°C denaturation. Lacks 3'→5' proofreading. Extends at ~1 kb/min. |
| Amplification formula | After n cycles, starting from 1 molecule: 2^n copies. After 30 cycles: ~1 billion copies. |
| Required reagents | Template DNA, forward primer, reverse primer, Taq polymerase, dNTPs (dATP, dCTP, dGTP, dTTP), MgCl2 buffer. |
| Applications | Forensics (STR-PCR), medical diagnosis (viral load), prenatal testing, evolutionary biology, GMO detection, RT-PCR for RNA viruses. |
| Common errors | Low annealing temperature → non-specific bands. Insufficient extension time → incomplete products. No template → no band. Contamination → false positives. |
SUMMARY BOX: PCR: Denaturation (94°C) → Annealing (50-65°C) → Extension (72°C) | Repeated 25-35 cycles | 2^n amplification | Taq from Thermus aquaticus | Key reagents: template + 2 primers + Taq + dNTPs