Skip to content
Login Register

#self-ligation

2 public questions tagged with this topic.

RDT Basics

Which enzyme prevents self-ligation of plasmid DNA?

Alkaline phosphatase hydrolyzes 5'-phosphomonoester bonds, converting 5'-phosphate termini of linearized plasmid DNA into 5'-hydroxyl groups. DNA ligase strictly requires a 5'-phosphate and adjacent 3'-hydroxyl to catalyze phosphodiester bond formation, so dephosphorylated vector molecules cannot self-circularize, thereby suppressing empty vector background. In cloning, the foreign insert retains chemically intact 5'-phosphates supplied by restriction digestion or polynucleotide kinase, enabling ligation at each junction to form a heteroduplex with two nicks that are repaired after transformation by bacterial repair machinery, greatly improving recombinant recovery.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details
Gene Cloning Process

Which enzyme is used to prevent self-ligation of a vector during cloning?

Alkaline phosphatase dephosphorylates the 5' ends of cleaved vector DNA, removing the phosphate groups essential for phosphodiester bond formation by DNA ligase. Self-ligation requires both 5' phosphate and 3' hydroxyl ends; dephosphorylation prevents recircularization of empty vector without insert. This forces ligation to depend on phosphate contributed by foreign DNA insert, greatly increasing recombinant yield and reducing background colonies. Calf intestinal phosphatase and shrimp alkaline phosphatase are commonly used, followed by heat inactivation or column purification before ligation and bacterial transformation steps.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

View Details