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BI10.1-7 | Molecular Biology — SDL Guide (Part 4)

PCR, Microarray, FISH, and CRISPR

Polymerase Chain Reaction (PCR):
Invented by Kary Mullis (1983), PCR amplifies a specific DNA segment millions of times in hours. The three steps repeat in cycles:
1. Denaturation (94°C) — DNA strands separate
2. Annealing (55-65°C) — primers bind to target sequences
3. Extension (72°C) — Taq polymerase (heat-stable, from Thermus aquaticus) synthesises new strands

After 30 cycles, one DNA molecule becomes ~1 billion copies. Clinical uses: COVID-19 RT-PCR testing, HIV viral load, TB diagnosis (GeneXpert), forensic DNA fingerprinting, prenatal genetic testing.

Microarray (DNA chip):
Thousands of known DNA sequences are fixed on a glass slide. Patient DNA or RNA is labelled with fluorescent dyes and hybridised to the chip. The pattern of fluorescence reveals which genes are active or mutated. Used for:
- Cancer gene expression profiling (e.g., classifying breast cancer subtypes)
- Pharmacogenomics (predicting drug response)

FISH (Fluorescence In Situ Hybridisation):
Fluorescent DNA probes bind to specific chromosomal locations, visible under a fluorescence microscope. Used to detect:
- Chromosomal translocations (e.g., Philadelphia chromosome in CML — BCR-ABL fusion)
- Gene deletions and duplications
- Trisomies (Down syndrome prenatal screening)

CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats):
A revolutionary gene editing tool borrowed from bacterial immune systems:
- A guide RNA directs the Cas9 enzyme to a specific DNA sequence
- Cas9 makes a precise double-strand cut
- The cell's repair machinery can then delete, correct, or insert DNA

CRISPR is being explored for:
- Sickle cell disease gene correction (clinical trials ongoing)
- Cancer immunotherapy (engineering T cells)
- Eliminating mosquito-borne diseases (gene drives)
- The 2020 Nobel Prize in Chemistry was awarded to Jennifer Doudna and Emmanuelle Charpentier for developing CRISPR-Cas9

Figure: PCR, Microarray, FISH, and CRISPR