Page 5 of 8

BI10.1-7 | Molecular Biology — Part 4

PCR, Microarray, FISH, and CRISPR

Molecular Diagnostic Techniques — Comparison

Technique	Principle	What It Detects	Key Clinical Uses
PCR	Thermal cycling amplification of specific DNA	Specific DNA/RNA sequences	COVID-19, HIV viral load, TB (GeneXpert), forensics
Microarray	Hybridisation of labeled DNA to probe array	Gene expression profiles, SNPs	Cancer classification, pharmacogenomics
FISH	Fluorescent probe hybridisation to chromosomes	Chromosomal translocations, amplifications, deletions	BCR-ABL in CML, HER2 in breast cancer, prenatal diagnosis
CRISPR-Cas9	Guide RNA-directed Cas9 endonuclease	Targeted gene editing	Sickle cell therapy, CAR-T cells, research knockout

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

PCR, Microarray, FISH, and CRISPR — Multi-panel illustration of molecular diagnostic techniques: PCR thermal cycling with exponential amplification, DNA microarray gene expression profiling, FISH for chromosomal translocations, and CRISPR-Cas9 gene editing mechanism with repair outcomes

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

CLINICAL PEARL

From bench to bedside in India: The Indian Council of Medical Research (ICMR) recommends GeneXpert (a PCR-based platform) as the first-line diagnostic test for tuberculosis. In 2023, India approved its first indigenous mRNA vaccine platform. CRISPR-based diagnostics (like the FELUDA test developed at CSIR-IGIB, Delhi) were deployed for rapid COVID-19 detection — a paper-based test delivering results in under an hour without expensive equipment. These technologies are transforming healthcare delivery in resource-limited settings.

SELF-CHECK — Molecular Technologies

A heat-stable DNA polymerase used in PCR is obtained from:

A. Escherichia coli

B. Thermus aquaticus

C. Saccharomyces cerevisiae

D. Staphylococcus aureus

Reveal Answer

Answer: B. Thermus aquaticus

FISH technique is commonly used to detect the Philadelphia chromosome in:

A. Acute lymphoblastic leukaemia

B. Chronic myeloid leukaemia

C. Hodgkin lymphoma

D. Multiple myeloma

Reveal Answer

Answer: B. Chronic myeloid leukaemia