Question

Describe ligation reaction. Discuss the possible products of ligation reaction. What happened to e coli that did not take any the vector? Drug resistance? What is the maximum transformation efficiency that can be expected? What things can influence the transformation efficiency and ligation reaction using 2 endonuclease for subcloning instead of one?

Answer 1

In molecular biology, ligation reaction is the joining of two fragments of DNA by the help of a ligase enzyme. The ligation occurs between the 3' hydroxyl group of one nucleotide from one fragment with the 5'-phosphoryl group of other nucleotide from second fragment. Generally the DNA fragments that are needed to be ligated in such a reaction are first joined with complimentary adapter polynucleotide sequences to ensure proper ligation and minimize the erroneous joining of the wrong fragments or fragments in wrong order.

In a cloning experiment, the DNA fragment which is cloned into a vector plasmid, are joined by ligation reaction. In absence of adapters, however, one can obtain different combinations of ligated products. For example, the vector plasmid ends can ligate together without the inclusion of the DNA fragment. Two or more DNA fragments can ligate together to form a spurious product. Two or more vectors can join with each other and form a high molecular weight product . Finally the plasmid and the DNA fragment can also ligate with each other to give a desired product that can be used to transform bacteria for cloning.

Generally, when transformed bacteria are selected to ensure efficient transformation, media are prepared with a specific antibiotic. The properly transformed bacteria that carry the vector can survive in the media due to the presence of antibiotic resistance gene present in the plasmid. So the E.coli. that did not take any vector would be susceptible to the antibiotic or drug that has been used to prepare the growth medium for the bacteria. These bacteria would be unable to survive.

Transformation efficiency is calculated by dividing the number of successful transformants by the amount of DNA used in the transformation procedure. Plasmid size, transformation protocol, DNA damage during transformation, all these affect the efficiency. Small plasmid with supercoiled DNA are good for transformation. Different strains of bacteria can have differential transforming ability e.g. E.coli K12 strain can use minimal media for growth and are preferred for transformation. Transformation protocols can also influence efficiency. These protocols are always subjected to standardization. Electroporation always give maximal efficiency for larger plasmids. Transformation efficiency is always determined in a scenario of excess viable cells that can be transformed so that availability of cells doesn't limit the procedure.

Theoretically 1x10¹¹ cfu/$\mu$g of DNA can be achieved. However, in practice , 2-4 x 10¹⁰ cfu/$\mu$g of DNA are achieved for a plasmid of standard size.

Endonucleases are enzymes that cleave the phosphodiester bonds between adjacent nucleotides. During cloning, endonucleases are used to cut the plasmid at a target sequence and then allowing ligation of the DNA fragment between the cut ends of the plasmid. Subcloning is a technique that involves the transfer of the DNA fragment from one plasmid to another plasmid. The endonuclease used to cut the DNA fragment and the second plasmid are same. This is done to produce complimentary sticky ends to facilitate ligation later. If two different endonucleases are used , the ends of the second plasmid and the DNA fragment will not be complimentary anymore and the ligation reaction would produce spurious products thereby decreasing the transformation efficiency.

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