8.4.1 Recombinant DNA Technology

1. Using Reverse Transcriptase to Make DNA

• Reverse transcriptase is an enzyme found in viruses and some bacteria.
• It catalyses the formation of complementary DNA (cDNA) from a single strand of RNA.
• mRNA is extracted from cells where the target gene is being expressed, and reverse transcriptase creates a working DNA copy of the gene.

2. Using Restriction Endonucleases to Cut DNA Fragments

• Restriction endonucleases are enzymes from bacteria that cut DNA at specific sequences (often 6 base pairs long).

• Enzymes that make staggered cuts produce sticky ends which are single-stranded overhangs.

• Sticky ends are complementary if the same restriction enzyme is used on both DNA fragments, allowing them to bind before forming stronger covalent bonds. Sticky Ends vs Blunt Ends:

• Sticky ends facilitate easier recombination compared to blunt ends (straight cuts).

3. In-Vivo Gene Cloning Using Vectors

• To protect inserted DNA from being degraded by enzymes, a vector is used.
• Plasmids from bacteria are common vectors.
• Steps:
  1. Cut plasmids and the target gene with the same restriction enzyme, creating complementary sticky ends.
  2. Mix fragments with plasmids to allow base pairing between sticky ends.
  3. Use DNA ligase to form strong phosphodiester bonds, creating recombinant DNA molecules.

4. Transformation of Host Cells

• Electroporation: Enhances uptake of plasmids by bacterial cells.
  • Bacteria are treated with calcium salts and subjected to rapid temperature changes (0°C → 40°C).
  • This increases membrane permeability, making it easier for the plasmid to enter.

5. Gene Markers

• Gene markers identify bacteria that successfully take up the plasmid or the desired gene.
• Types of gene markers: 4. Antibiotic resistance genes: Bacteria with plasmids survive when grown on antibiotic-containing media. 5. Fluorescent markers: Genes producing fluorescent proteins indicate plasmid uptake under UV light. 6. Enzyme markers: Bacteria with plasmids produce observable changes in colour when exposed to specific substrates. Key Note: Marker genes can confirm whether the plasmid contains the desired DNA, as the marker gene may be inactivated if the gene of interest is inserted into it.