Aquaculture & Marine Biotechnology

• Vector Dephosphorylation in Recombinant DNA Technology:
• When cutting plasmid cloning vectors with a single restriction endonuclease, complementary cohesive ends readily recircularize and self-ligate in the presence of DNA ligase, dramatically reducing recombinant cloning efficiency.
• Alkaline Phosphatase (Calf Intestinal Phosphatase [CIP], Shrimp Alkaline Phosphatase [SAP], or Antarctic Phosphatase):
• Catalyzes the hydrolytic removal of $5'$-terminal phosphate groups from linearized vector DNA.
• Because DNA ligase strictly requires a $5'$-phosphate and a $3'$-OH to catalyze phosphodiester bond formation, dephosphorylated vectors cannot self-ligate and can only incorporate insert fragments providing a $5'$-phosphate.
• nalysis:
• (Alkaline Phosphatase): Correct.

Correct option: (3) Alkaline Phosphatase

• Site-Specific Recombination:
• Recombination between specific short, defined DNA sequence motifs (attachment sites, loxP, FRT) catalyzed by dedicated recombinases.
• Integrase (Site-Specific Recombinase):
• Belonging to the tyrosine (e.g., Bacteriophage $\lambda$ Integrase, Cre recombinase, FLP recombinase) or serine recombinase families.
• Integrases recognize specific inverted repeats, cleave DNA strands, exchange homologous ends, and ligate them without requiring extensive homology or external ATP.
• nalysis:
• (Integrase): Correct.

Correct option: (3) Integrase

• Homologous Recombination Model:
• Proposed by Robin Holliday in 1964:
• Homologous recombination (meiotic crossing-over and double-strand break repair) proceeds through the invasion of a single strand into an intact homologous duplex, creating a cross-strand four-way DNA intermediate termed the Holliday junction.
• Branch migration extends the heteroduplex region, followed by resolution of the Holliday junction by junction-resolving endonucleases (resolvases like RuvC in E. coli or GEN1 in eukaryotes) in either horizontal or vertical planes, yielding crossover or non-crossover recombinants.
• nalysis:
• (Homologous recombination): Correct.

Correct option: (4) Homologous recombination