• Bacterial Restriction-Modification (R-M) System:
• Bacteria synthesize sequence-specific DNA methyltransferases that add methyl groups ($-CH_3$) to adenine or cytosine bases within their own genomic recognition motifs.
• This methylation prevents host restriction endonucleases from binding and cleaving the bacterial chromosome, protecting host genomic DNA from degradation by its own restriction enzymes while degrading unmethylated foreign bacteriophage DNA.
• Correct Option: Option 4 (Protect DNA from its own restriction enzymes).

• Alkaline Lysis Principle (Birnboim & Doly, 1979):
• During plasmid extraction, bacteria are lysed using a solution of $0.2\text{ N NaOH}$ and $1\%\text{ SDS}$, creating an alkaline pH ($\approx 12.0 - 12.5$).
• Denaturation Behavior:
• Chromosomal DNA: High alkalinity breaks hydrogen bonds between base pairs, causing large, sheared, linear chromosomal DNA strands to completely separate and denature irreversibly.
• Plasmid DNA: Small, covalently closed circular (ccc) plasmids denature partially but remain topologically intertwined.
• Neutralization: Rapid neutralization with acidic potassium acetate ($pH \approx 4.8 - 5.2$) allows plasmid circles to renature correctly, whereas genomic DNA tangles irreversibly into an insoluble aggregate that precipitates with SDS-protein complexes.
• nalysis:
• (Alkalinity; irreversibly): Correct.

Correct option: (2) Alkalinity; irreversibly

• 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