Recombinant DNA Technology and Genetic Engineering
What the domain covers
Recombinant DNA technology and genetic engineering tests the tools and steps used to move and express DNA: restriction enzymes, ligase, vectors, hosts, transformation, selection, screening, PCR, electrophoresis, and cloning. Expect questions on enzyme function, plasmid design, and interpreting cloning results.
Core rules and concepts
- Restriction endonucleases cut DNA at specific palindromic recognition sites, producing sticky or blunt ends. Ligase joins compatible ends by forming phosphodiester bonds.
- A vector must replicate in the host, carry a selectable marker, and have a cloning site. Plasmids, bacteriophages, cosmids, BACs, and expression vectors serve different insert sizes and purposes.
- Transformation introduces DNA into competent cells; selection uses a marker such as ampicillin resistance; screening distinguishes recombinants from non-recombinants.
- PCR uses template, primers, dNTPs, thermostable DNA polymerase, and buffer, cycling through denaturation, annealing, and extension.
- Agarose gel electrophoresis separates DNA by size; smaller fragments migrate farther.
Worked example
A gene is inserted into the multiple cloning site of pUC19, inside lacZ. Bacteria are plated on ampicillin plus X-gal. Cells taking up plasmid grow; colonies with an intact lacZ make blue product, while colonies with an insert have disrupted lacZ and appear white. Reasoning: ampicillin selection confirms plasmid uptake; blue-white screening detects insert presence, not expression of the target gene.
Traps
Do not say ligase cuts DNA or that restriction enzymes join ends. Do not confuse selection with screening: antibiotic resistance selects transformants; blue-white or colony PCR screens for recombinants. Do not assume every white colony has the correct insert; confirm by sequencing or restriction mapping.