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#vector preparation

2 public questions tagged with this topic.

Gene Cloning Process

Why is alkaline phosphatase used in cloning?

DNA ligase catalyzes phosphodiester bond formation only between adjacent 3' hydroxyl and 5' phosphate termini. Vector linearized with single restriction enzyme retains complementary sticky ends bearing 5' phosphates, readily self-ligating and producing high background of empty clones. Alkaline phosphatase, including calf intestinal or shrimp isoforms, hydrolyzes 5' phosphate groups leaving 5' hydroxyl termini, rendering the vector ligation-incompetent unless paired with phosphorylated insert DNA supplying phosphates. Subsequent ligation yields circular recombinant product with one nick at each junction, later repaired after transformation into host bacteria.

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.

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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.

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