⚡ Overview: Module 5 – Recombinant DNA Technology & Genetic Engineering
Genetic engineering involves manipulating genetic material (DNA and RNA) in vitro to alter the phenotype of host organisms. The central paradigm relies on cutting DNA with restriction endonucleases, joining fragments via DNA ligase, inserting chimeric constructs into cloning vectors, and introducing them into competent host cells for propagation and expression.
Core Topics in this Module
Topic 1
Polymerase Chain Reaction (PCR)
Denaturation, primer annealing, and thermostable Taq polymerase extension. RT-PCR and real-time quantitative qPCR.
Topic 2
DNA Sequencing
Sanger dideoxy chain-termination chemistry (ddNTPs), capillary electropherograms, Next-Generation Sequencing (NGS), and Nanopore.
Topic 3
CRISPR-Cas9 & Gene Editing
Synthetic sgRNA guidance, PAM sequence recognition (5′-NGG-3′), targeted double-strand breaks, NHEJ knockout, and HDR knock-in.
Key Tools of Recombinant DNA Technology
- Restriction Enzymes (Molecular Scissors): Type II restriction endonucleases recognize specific palindromic sequences (e.g., EcoRI recognizes 5′-GAATTC-3′) and cleave phosphodiester backbones, leaving complementary staggered “sticky ends”.
- Cloning Vectors (Molecular Vehicles): Must possess an Origin of Replication (ori), selectable marker genes (e.g., ampicillin/tetracycline resistance), and a Multiple Cloning Site (MCS / polylinker). Examples include plasmids (pBR322, pUC19), bacteriophages (λ phage), cosmids, BACs, and YACs.