Recombinant DNA Technology and Gene Cloning

Applications of Recombinant DNA Technology

Gene Cloning: Production of many identical copies of the target DNA.
Types of Gene Cloning:
- In Vivo: Involves living organisms. Example: Bacteria used as host cells.
- In Vitro: Performed outside living organisms. Example: PCR (Polymerase Chain Reaction).

There are five main steps:

Isolation
- Target DNA & Cloning Vector: Plasmids are extracted from bacterial cells.
- Example of Target DNA: Eye color gene from a human chromosome.
Cutting
- Use of restriction enzymes to cut plasmid and target DNA at specific sites.
- Restriction Enzymes: ECO R1 cuts at the site GAA-TTC producing "staggered" cuts.
- MCS (Multiple Cloning Site): Part of the plasmid containing restriction sites.
Insertion
- Inserting target DNA into the plasmid MCS.
- Using ligase enzymes to form a phosphodiester bond between target DNA and plasmid.
Transformation and Amplification
- Transformation: Introducing recombinant DNA into the host cells (bacteria).
- Amplification: Replication of recombinant plasmid and division of host cells through DNA replication and binary fission.
Screening
- Identifying host cells containing the plasmid with the target DNA.
- Blue-White Screening Process:
  - Blue Colonies: Host cells with plasmid but without target DNA.
  - White Colonies: Host cells with plasmid containing target DNA.
  - Like Z Gene: Disrupted by target DNA insertion, so no beta-galactosidase enzyme is produced.
  - Ampicillin Resistance Gene (AMPR): Allows bacteria to survive in antibiotics.

Plasmid Components:
- Ori Sequence: For plasmid replication.
- MCS: Multiple cloning site.
- Selectable Markers: Lexed gene and antibiotic resistance gene.
Function of Lexed Gene: Produces beta-galactosidase enzyme that hydrolyzes X-Gal into X and galactose. X produces a blue color.
Antibiotic Resistant Gene: Allows bacterial cells to survive in antibiotic medium.