Question
PCR – Principle
Answer
PCR (Polymerase Chain Reaction) — Principle
PCR is an in-vitro molecular technique used to exponentially amplify a specific, targeted segment of DNA, generating millions of copies from a very small starting quantity, enabling sensitive detection/analysis of that DNA sequence.
Components: template DNA (containing the target sequence); a pair of primers (short, sequence-specific synthetic oligonucleotides flanking the target region); thermostable DNA polymerase (classically Taq polymerase, derived from the thermophilic bacterium Thermus aquaticus, able to withstand repeated high-temperature cycles without denaturing); deoxynucleotide triphosphates (dNTPs) — the building blocks for new DNA strand synthesis; and a suitable buffer with Mg²⁺.
Principle — three repeating steps per cycle:
- Denaturation (~94–96°C) — the double-stranded template DNA is heated, separating it into two single strands.
- Annealing (~50–65°C, primer-specific) — the temperature is lowered, allowing the two primers to bind (hybridize) specifically to their complementary sequences flanking the target region on each single strand.
- Extension (~72°C, optimal for Taq polymerase) — the thermostable DNA polymerase extends each primer, synthesizing a new complementary DNA strand using the available dNTPs.
This three-step cycle is repeated 25–35 times in an automated thermal cycler; since each cycle doubles the amount of target DNA, the target sequence is amplified exponentially (2^n, where n = number of cycles), producing enough copies for detection (typically by gel electrophoresis, or in real-time PCR, by fluorescent probe/dye signal monitored during amplification).
Applications: diagnosis of infectious diseases (detecting pathogen-specific DNA/RNA — after reverse transcription for RNA targets, RT-PCR); genetic disease diagnosis; forensic DNA analysis; detection of antibiotic-resistance genes; research applications (cloning, sequencing preparation).