Biotechnology and Its Applications
Domain 9 covers recombinant DNA technology and its applications. Questions test restriction enzymes, vectors, PCR, gel electrophoresis, transgenic organisms, gene therapy and biosafety.
Core rules
- Recombinant DNA uses restriction endonucleases to cut DNA at specific palindromic sequences, DNA ligase to join fragments, and vectors such as plasmids to carry DNA into host cells.
- A good vector has an origin of replication, selectable marker and unique restriction sites. Agrobacterium tumefaciens Ti plasmid is used in plant transformation.
- PCR amplifies DNA using denaturation, primer annealing and extension by Taq polymerase.
- Gel electrophoresis separates DNA by size; smaller fragments move faster.
- Applications include insulin production, Bt cotton, golden rice, gene therapy and RNA interference. Bt toxin is activated in the alkaline gut of insects.
Worked example
A human insulin gene is inserted into a plasmid. Explain the sequence. Cut the gene and plasmid with the same restriction enzyme to create compatible ends; join with DNA ligase; transform E. coli; select transformants using the marker; confirm insert and expression. Reasoning: same enzyme prevents mismatched ends, ligase forms phosphodiester bonds, and the marker identifies cells that took up the plasmid.
Traps
- Saying restriction enzymes cut randomly; they cut specific recognition sites.
- Confusing DNA ligase with DNA polymerase; ligase joins, polymerase synthesises.
- Claiming Bt toxin kills all insects; it is specific after activation in susceptible insect guts.
- Treating gene therapy as changing all body cells; somatic therapy affects only the patient.