Genetic Engineering and Recombinant DNA Technology
This section covers the tools and workflow of recombinant DNA technology. The exam tests restriction enzymes, vectors, ligation, transformation, selection, screening, and PCR.
Core workflow
A target gene and a vector are cut with the same restriction enzyme or with enzymes that leave compatible ends. EcoRI recognizes 5-prime GAATTC and cuts between G and A, leaving a 5-prime AATT sticky end. DNA ligase seals the sugar-phosphate backbone. The recombinant plasmid is introduced into a host such as E. coli by transformation. Antibiotic resistance markers select cells that took up a plasmid. Screening methods include blue-white selection, colony PCR, restriction mapping, and sequencing. PCR uses denaturation near 94-95 degrees Celsius, primer annealing around 50-65 degrees Celsius, and extension near 72 degrees Celsius with Taq polymerase. Vectors include plasmids, bacteriophages, cosmids, BACs, and expression vectors.
Worked example
An insert has EcoRI sites at both ends and the plasmid has an EcoRI site plus an ampicillin-resistance gene. Digest both with EcoRI, ligate, transform, and plate on ampicillin. Only plasmid-bearing cells grow. If the plasmid also carries lacZ and the insert disrupts it, recombinant colonies are white on X-gal, while non-recombinant colonies are blue. Antibiotic selection alone does not prove the insert is present.
Traps that cost marks
Do not use different, incompatible enzymes and expect ligation. Antibiotic selection selects for the vector, not automatically for the insert. Restriction enzymes do not cut randomly; they recognize specific sequences. PCR requires primers, and ordinary Taq lacks proofreading. Do not confuse transformation with transfection.