D1.1.4 Polymerase chain reaction and gel electrophoresis Notes

Polymerase Chain Reaction (PCR): Amplifying DNA

This allows scientists to study tiny DNA samples from crime scenes, fossils, or other sources.

Key Components of PCR

1. Template DNA: The DNA you want to copy.
2. Primers: Short DNA sequences that bind to the template, marking the starting point for replication.
3. Taq Polymerase: A heat-resistant enzyme that synthesizes new DNA strands.
4. Nucleotides (dNTPs): The building blocks of DNA.
5. Thermal Cycler: A machine that precisely controls temperature changes.

Steps of PCR (DNA Amplification)

PCR consists of a cycle of three main steps, repeated 20–40 times to exponentially amplify the target DNA sequence.

1. Sample Preparation
   1. The DNA sample is extracted and purified.
   2. Template DNA (the DNA to be copied) is prepared along with other PCR components.
2. Denaturation (~94-96°C) → Fragmentation
   1. The DNA is heated to ~95°C.
   2. This breaks the hydrogen bonds between complementary base pairs, separating the double-stranded DNA into two single strands.
   3. These single strands serve as templates for new DNA synthesis.
3. Annealing (~50-65°C) → Primer Annealing
   1. The temperature is lowered to 50–65°C.
   2. Short DNA sequences called primers bind (anneal) to complementary sequences on each single-stranded DNA template.
   3. Primers mark the starting point for DNA synthesis and are essential because Taq polymerase cannot start building DNA without them.
4. Extension (~72°C) → DNA Synthesis
   1. The temperature is raised to 72°C, the optimal temperature for Taq polymerase.
   2. Taq polymerase is a heat-resistant enzyme that adds nucleotides (dNTPs) to the primers, synthesizing new DNA strands in the 5' to 3' direction.
   3. After one cycle, the amount of DNA doubles.
5. Separation and Data Analysis → Gel Electrophoresis
   1. Once DNA is amplified by PCR, gel electrophoresis is used to separate and analyze the DNA fragments.

Why Taq Polymerase?

1. Taq polymerase remains stable at high temperatures (~95°C), unlike most enzymes that would denature.
2. This allows it to survive the repeated heating cycles in PCR.

Gel Electrophoresis: Separating DNA Fragments by Size

How Gel Electrophoresis Works

1. Preparation of the gel: A gel (usually agarose) is poured into a mold with wells at one end.
2. Loading the DNA: DNA samples are mixed with a dye and loaded into the wells.
3. Applying an electric field: The gel is placed in a buffer solution, and an electric current is applied. • The negative electrode is near the wells. • The positive electrode is at the opposite end.
4. Migration of DNA: DNA is negatively charged (due to its phosphate backbone), so it moves toward the positive electrode. • Smaller fragments travel faster and farther through the gel. • Larger fragments move slower.
5. Visualization: The gel is stained with a dye (e.g., ethidium bromide) that binds to DNA, making fragments visible under UV light.

Why DNA Separates By Size

1. The gel acts like a sieve, slowing down larger fragments more than smaller ones.
2. This allows DNA fragments to be separated and analyzed based on their size.