Ligation and ligases

Question 1

What are DNA ligases?

Answer 1

DNA ligases are enzymes that holds the ends of two DNA strands together by forming a phosphodiester bond between two DNA ends.

Question 2

State two of the most important ligases and their cofactor.

Answer 2

• Bacteriophage T4 Ligase (ATP cofactor)

- E. coli DNA ligase (NAD+ cofactor)

Question 3

What are the biological functions of DNA ligases?

Answer 3

• DNA repair
• Synthesis of lagging strand
• Genetic recombination

Question 4

Describe the mechanism of ligase action.

Answer 4

• Covalent enzyme-AMP intermediate is formed on the lysine side chain of ligase.
• The AMP nucleotide is transferred to the 5’ end of the nicked DNA strand.
• The 3’ OH group of the nicked DNA attacks the AMP-DNA bond, sealing the phosphate backbone and resealing AMP.

Question 5

Why is “sticky” end ligation more useful in molecular biology?

Answer 5

The hydrogen bonding of cohesive ends increases the efficiency of cohesive-end ligation.

Question 5

Why is “sticky” end ligation more useful in molecular biology?

Answer 5

The hydrogen bonding of cohesive ends increases the efficiency of cohesive-end ligation.

Question 6

What are two common vector:insert ratio that is used?

Answer 6

• 1:1 and 1:3 vector:insert