🧬 Genetic Engineering: Recombinant DNA & Cloning Vectors
Recombinant DNA technology combines genetic sequences from heterologous organisms using restriction endonucleases, DNA ligases, and cloning vectors for gene amplification, expression, and functional characterization.
1. Palindromic Restriction Endonucleases
Type II restriction enzymes recognize symmetric palindromic sequences (4–8 bp) and cleave phosphodiester backbones in the presence of $Mg^{2+}$ without requiring ATP:
| Enzyme | Source Organism | Recognition Sequence & Cut ($/$) | Cleavage End Geometry |
|---|---|---|---|
| EcoRI | Escherichia coli RY13 | $5’text{-G / A A T T C-3′}$ | $5’$ sticky overhang ($4text{ nt}$) |
| BamHI | Bacillus amyloliquefaciens H | $5’text{-G / G A T C C-3′}$ | $5’$ sticky overhang ($4text{ nt}$) |
| HindIII | Haemophilus influenzae Rd | $5’text{-A / A G C T T-3′}$ | $5’$ sticky overhang ($4text{ nt}$) |
| SmaI | Serratia marcescens | $5’text{-C C C / G G G-3′}$ | Blunt end (no overhang) |
| PstI | Providencia stuartii | $5’text{-C T G C A / G-3′}$ | $3’$ sticky overhang ($4text{ nt}$) |
2. Essential Features of Modern Plasmid Cloning Vectors
- Origin of Replication (ori): Autonomous replication cassette (e.g. pMB1 or ColE1 origin yielding 300–500 copies/cell).
- Selectable Marker Gene: Antibiotic resistance gene (e.g. $beta$-lactamase conferring Ampicillin resistance; $kan^R$ for Kanamycin).
- Multiple Cloning Site (MCS / Polylinker): Synthetic cluster of 15–25 unique restriction enzyme recognition sites located within the 5′ region of the lacZ’ gene.
- $alpha$-Complementation (Blue/White Screening): Insertion of foreign DNA into the MCS disrupts the reading frame of $beta$-galactosidase $alpha$-peptide. Recombinants remain white on X-gal + IPTG agar plates, whereas non-recombinants produce active enzyme and convert X-gal into 5,5′-dibromo-4,4′-dichloro-indigo (blue colonies).
