Discuss the following questions with your partner and be prepared to share your

Question

Discuss the following questions with your partner and be prepared to share your answers with the class:

1. What role do DNA ligases have in nature?

2. What role do DNA ligases have in gene cloning?

3. What properties of the DNA restriction fragments produced in Laboratory 2 enable ligation of these fragments?

4. Could two rfp fragments join to form a plasmid during the ligation? If not, what would prevent that? If so, what would be the result?

5. During ligation, both hydrogen and covalent bonds form. Which bonds form first? Why do both types of bonds need to form?

Explanation / Answer

1.

DNA replication in the lagging strand: The lagging strand receives several RNA primers. Replication process in the lagging strands is more complicated due to its orientation (its direction of replication is opposite to that of growing replication fork).

The DNA template of lagging strand is read by a primase, which synthesise complementary RNA primer. The primed segments are extended by DNA polymerase forming the Okazaki fragments, now the RNA primers are removed. The synthesis of lagging stand occurs using short DNA segments called Okazaki fragments each consisting of 100 to 200 nucleotides in humans (1000 to 2000 nucleotides in bacteria). The replication occurs in 5 to 3 direction, but away from the replication fork.  DNA ligase connects the Okazaki fragments by catalysing the formation of phosphodiester bond between the Okazaki fragments of complementary strand.

DNA ligase also seals the DNA during repair process.

2. During coning, the vector is physically joined to a particular region of gene of interest. DNA ligase is necessary to seal these joints.

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