Recombinant DNA and Genetic Engineering
Recombinant DNA and Genetic Engineering tests cloning design, vector choice, transformation, selection, screening, and regulated editing.
Core rules
- Restriction endonucleases cut specific palindromic sequences, often leaving sticky ends. Ligase joins compatible ends; alkaline phosphatase reduces vector self-ligation.
- Plasmids carry an origin of replication, selectable marker, and multiple cloning site. Expression vectors add a promoter and ribosome-binding site.
- Transformation introduces DNA into competent cells; selection uses an antibiotic marker, but selection does not prove the insert is correct.
- Screening may use colony PCR, restriction digest, blue-white screening, or sequencing. PCR contamination and primer dimers are common false positives.
- CRISPR-Cas9 creates targeted double-strand breaks; repair by NHEJ can knock out a gene, while homology-directed repair can insert a sequence.
- Containment and approval are required for genetically modified organisms.
Worked example
A gene is amplified with primers carrying BamHI sites, digested, and ligated into a BamHI-cut plasmid. The vector is dephosphorylated to reduce self-ligation. E. coli is transformed and plated on ampicillin. Colonies are screened by colony PCR using one primer in the insert and one in the vector. A colony that grows on ampicillin but gives no PCR product may contain empty vector, so antibiotic resistance alone is insufficient.
Traps
Do not assume a restriction enzyme cuts only once; check the vector map. Do not use the same antibiotic marker for cloning and expression without checking compatibility. Do not confuse transformation with transfection. Do not treat a PCR band as sequence proof. Do not ignore off-target effects in CRISPR experiments.