Biotechnology and Recombinant DNA Technology

Introduction

• Lecturer: Andy from Med School EU
• Topics: Biotechnology, Recombinant DNA Technology, and Applications

Polymerase Chain Reaction (PCR)

• Definition: Amplification of a gene into large quantities.
• Purpose: Detect pathogens or specific genes (e.g., used in COVID tests).
• Process:
  1. Heating: Separate DNA strands by breaking hydrogen bonds.
  2. Adding Primers: Mark specific DNA sequences.
  3. Adding Taq DNA Polymerase:
     • Thermostable to function at high temperatures.
     • Derived from Thermus aquaticus bacterium.
  4. Cycle Repetition: Typically 25 to 40 cycles.
• Requirements: Magnesium, dNTPs, template DNA, primers.

Gene Cloning Using Bacteria

• Purpose: To clone specific genes (e.g., insulin gene) using bacterial plasmids.
• Steps:
  1. Isolate gene of interest using restriction enzymes.
  2. Use restriction enzymes on bacterial plasmid.
  3. Combine gene of interest with bacterial plasmid (acts as a vector).
  4. Use DNA ligase to seal fragments.
• Vectors:
  • Types: Plasmids, Viral vectors, Cosmids, Artificial chromosomes.
  • Most common: Bacterial plasmid.

Screening for Recombinant Plasmids

• Process:
  1. Transform bacteria with plasmids.
  2. Grow on medium containing ampicillin and X-gal.
  3. Identify recombinant (white colonies) vs. non-recombinant (blue colonies) plasmids.

Reverse Transcription

• Definition: Conversion of RNA back to DNA.
• Enzyme: Reverse transcriptase.
• Process:
  1. Use RNA template with a primer.
  2. Enzyme synthesizes complementary DNA (cDNA).
• Application: RNA viruses, retroviruses like HIV.

Digestion and Ligation of Restriction Fragments

• Restriction Enzymes:
  • Cut DNA at specific sites, creating sticky or blunt ends.
• Ligation:
  • DNA ligase seals sticky ends back together.
  • Important for gene cloning.

Conclusion

• End of lectures on reproduction and inheritance in biotechnology.
• Next topic: Inheritance and the environment focusing on mutations.