Source: Wikimedia Commons — Recombinant DNA technology overview
| CUE COLUMN | NOTE-TAKING COLUMN |
|---|---|
| What is rDNA technology? | Recombinant DNA (rDNA) technology combines DNA from two or more sources to create a new DNA molecule not found in nature. It uses restriction enzymes (molecular scissors), DNA ligase (molecular glue), and vectors (DNA vehicles) to insert foreign genes into host cells. |
| What is a restriction enzyme? | Enzymes from bacteria that recognize specific 4-8 bp palindromic sequences and cut the DNA at or near that site. EcoRI (from E. coli RY13) recognizes 5'-GAATTC-3' and produces 4-nt sticky ends (5'-AATT overhang). Over 900 enzymes identified from 230+ bacterial strains. |
| What are sticky vs. blunt ends? | Sticky ends = staggered cuts leaving ssDNA overhangs (EcoRI). Blunt ends = straight cuts with no overhangs (SmaI, EcoRV). Sticky ends preferred in cloning — complementary ends hybridize specifically for efficient ligation. |
| What is pBR322? | Classic E. coli cloning vector with 4 features: ori (replication), ampR (ampicillin resistance), tetR (tetracycline resistance), restriction sites. Recombinants identified by insertional inactivation (insert disrupts tetR). |
| What is the Ti plasmid? | Tumor-inducing plasmid from Agrobacterium tumefaciens. T-DNA integrates into plant chromosomes. Disarmed (tumor genes removed) for plant genetic engineering. Best for dicots; monocots need biolistics. |
| What is PCR? | In vitro DNA amplification using thermal cycling. Kary Mullis (Nobel 1993). Steps: Denaturation (94°C) → Annealing (50-65°C) → Extension (72°C). Taq polymerase (from Thermus aquaticus) is thermostable. After n cycles: 2^n copies. |
| What is gel electrophoresis? | Separates DNA by size through agarose gel. DNA (negatively charged from phosphate backbone) migrates to anode (+). Smaller fragments travel farther. Stained with ethidium bromide; visualized under UV as orange bands. |
| What is a bioreactor? | Large-scale vessel for producing biological products. Stirred-tank type has agitator (mixing), sparger (air supply), and sensors for temperature/pH/O2 control. Followed by downstream processing (separation → purification → formulation → QC). |
| How is DNA isolated? | Cell lysis (lysozyme for bacteria, cellulase for plants) → RNA removal (RNase) → Protein removal (protease) → Ethanol precipitation (DNA spools as threads). |
| How are cells transformed? | CaCl2 + heat shock (42°C, 60-90 sec) for bacteria. Also: microinjection (into nucleus), biolistics , electroporation (electrical pulses create pores). |
SUMMARY BOX: Biotechnology (rDNA technology) uses restriction enzymes to cut DNA at palindromic sites, DNA ligase to join fragments into vectors, and transformation methods to introduce recombinant DNA into host cells. PCR amplifies specific DNA sequences in vitro. Gel electrophoresis separates DNA by size. Bioreactors scale up production.