PCR

Polyermase Chain Reaction (PCR) is a technique used to make many copies of a specific segment of DNA. It allows scientists to amplify a target region from a very small starting amount of DNA.

How it works:

PCR takes place in three main steps that repeat for tens of (usually 25-35, but it varies) cycles:

1. Denaturation: The reaction is heated to around 95°C to break the hydrogen bonds between the two strands of DNA. This separates the double-stranded DNA into single strands.

2. Annealing: The temperature is lowered to around 50–65°C to allow short primers to bind to the complementary sequences flanking the target DNA. These primers are designed to match the region of interest.

3. Extension: The temperature is raised to about 72°C, the optimal temperature for the enzyme Taq polymerase. This enzyme adds nucleotides to the primers, building new DNA strands that are complementary to the original templates.

Each cycle doubles the amount of target DNA, resulting in millions of copies by the end of the process.

PCR relies on a series of heating and cooling cycles. First, the DNA is heated to separate the strands. Then, short primers bind to the specific regions flanking the target sequence. A heat-stable enzyme called Taq polymerase adds nucleotides to extend from the primers, copying the target DNA. This cycle repeats, and the DNA doubles with each round. After about 30–40 cycles, millions of copies of the target DNA have been made.

Applications:

• Detecting genetic mutations or infectious agents (e.g., in COVID-19 testing)

• Forensic analysis and DNA fingerprinting

• Analyzing gene expression (when combined with reverse transcription)

• Cloning and gene editing experiments

How to interpret data:

PCR products are often visualized using gel electrophoresis. A band of the expected size indicates that the target DNA was successfully amplified. Quantitative PCR (qPCR) uses fluorescent markers to measure the amount of DNA produced in real time.