Question

Explain the steps involved in making cDNA from mRNA, and how you would create a cDNA library. What type of vector would you use, and what steps would you need to take to prepare the cDNA for ligation with the vector?

Answer 1

Complementary DNA or cDNA is synthesized from a messenger RNA or mRNA with the help of an enzyme called reverse transcriptase along with another called DNA polymerase.

The first step in the cDNA preparaton is mRNA purification. This is done by several methods which include triazol extraction and column purification. The seperated mRNA can be eluted out using eluting buffer.

Once the mRNA has been seperated and purified, oligodeoxynucletides are added to the poly-A tail which acts as a complementary primer. Now the poly-A tail provides free OH-end, this is extended by the reverse transcriptase enzyme. This is hoe cDNA strand is created. The RNA strand is removed then by adding RNase which chews up the RNA strance leaving the cDNA strand. Now this is a single stranded cDNA (sscDNA) which needs to complementarily base paired which is done by the enzyme DNA Polymerase enzyme. The free OH group fror the DNA polymerase to synthesize the complementary strand is provided by the sscDNA itself by generating a hairpin loop at the 3' end by coiling on itself.

A cDNA library consist of cDNA fragments inserted into a variety of host cells . cDNA library consist of only cDNA of expressed genes.

The vector used depends upon the host cells and the type of cDNA prepared.