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 ligases join 5’ and 3’ends of newly synthesized DNA strands by catalysing the formation of phosphodiester bond. They work during DNA repair mechanism also. They play a role in repairing single-stranded breaks in duplex DNA. The DNA ligase IV specifically repairs double-strand breaks.

2.

In gene cloning we use plasmids. When we digest plasmids, for example if we cut the plasmid at certain base pairs, ligase is the enzyme that pastes these cut ends together to make a whole recombinant DNA plasmid again.

3.

The property that both plasmids were cut or digested with the same restriction enzymes, BamH-I and Hind-III has resulted in sticky ends that are complementary.

4.

Yes, two rfp fragments can join to form a plasmid during the ligation. The plasmid will make new parts and form recombinant DNA.

5.

Hydrogen bonds form first. Both types of bonds are needed. Hydrogen bonds form first between complementary bases and then covalent bonds form between sugars and phosphate groups.

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