PCR in Molecular Biology

Analysis of DNA - at the Gene Level

Polymerase Chain Reaction (PCR)

• PCR is a sensitive technique that can amplify a specific segment of DNA from a minute amount of template DNA.
• It requires two specific primers that bind specifically to a known sequence on the template DNA.
• The PCR cycle consists of three steps: denaturation, renaturation (annealing), and DNA synthesis.
• The cycle is repeated for 25-35 cycles, resulting in an exponential increase in the amount of target DNA (2n, where n is the number of cycles).
• PCR is limited to amplifying segments of DNA up to 1000 bp.

DNA Sequencing

• DNA sequencing is the ultimate analytical assay for determining the individual nucleotides that make up a DNA molecule.
• The Sanger dideoxy chain termination method was established in 1975.
• DNA molecules are separated in a gel and migrate due to the presence of an electric charge.
• DNA fragments are separated according to size, with smaller fragments migrating quickly and larger fragments migrating more slowly.
• The separated DNA can be visualized under UV light by staining with ethidium bromide.

Gel Electrophoresis

• Gel electrophoresis is a technique that separates macromolecules (proteins/DNA/RNA) on the basis of their size or charge.
• DNA molecules are negatively charged and move towards the positively charged electrode when placed in an electric field.
• DNA fragments can be separated according to size, with smaller fragments migrating quickly and larger fragments migrating more slowly.

Southern Blotting / Hybridisation

• Southern blotting is a technique where DNA separated by electrophoresis is transferred to a membrane filter and detected by the binding of a labelled probe.
• The technique is used to detect variations in large DNA fragments.
• The steps involved in Southern blotting are: DNA molecule cutting with RE, separation of DNA fragments on a gel, denaturation of DNA into single strands, transfer of DNA to a membrane filter, and hybridization with a labelled probe.
• Hybridization involves adding a labelled probe that binds to specific complementary DNA sequences on the membrane.

Restriction Analysis / Gene Cloning

• Restriction enzymes recognize and cut specific sequences of DNA (restriction sites) to create single-stranded ends.
• The single-stranded tails are "sticky" and can form hydrogen bonds with complementary sticky tails on other DNA molecules cut with the same enzyme.
• The gaps between the fragments are closed by DNA ligase, creating a single recombinant DNA molecule.
• Restriction enzymes are crucial tools in many molecular biological techniques, such as gene cloning.
• Gene cloning involves introducing a foreign DNA fragment into an organism to create a population of organisms with the same gene or genes.
• There are over 600 commercially available restriction enzymes.

This quiz covers the basics of Polymerase Chain Reaction (PCR), a laboratory technique used to amplify DNA sequences. It discusses the application of PCR in gene analysis, disease diagnosis, and detection of pathogens.