Question

Describe the 5 events that convert an mRNA into a cDNA.

Answer 1

Conversion of mRNA to cDNA involve following steps which are as follows....

1. Isolation of mRNA :- A family of mRNA is present in the cells and tissues. It is isolated from the cells.

2. Reverse transcription :- The family of mRNAs in solution is treated with oligo d (T) primer , the enzyme reverse transcriptase and four types of deoxyribonucleotides. The temperature is maintained at 55 degree centrigrade which is suitable for polymerization.

The oligo d (T) primer gets bound with poly A tail of the eukaryotic mRNAs. The primer provides a free 3'-OH group to proceed the reverse transcription.

Reverse transcriptase adds complementary deoxyribonucleotides one by one to the 3'-OH group of the primer and forms a single stranded DNA. As a result an RNA-DNA hybrid is formed. The single stranded DNA which is complementary to the mRNA is called complementary DNA (cDNA).

3. Oligo d(C) Tailing :- After forming RNA-DNA hybrid , it is treated with the enzyme terminal transferase and nucleotided CTP. This enzyme adds CTP one by one to the 3'-OH group of both the RNA and DNA strands. As a result , a short oligo d(C) tail is formed at the 3' end of both the strands.

4. Alkaline hydrolysis of mRNA :- The reaction mixture is treated with alkaline sucrose solution. The alkaline solution breaks hydrogen bonds between the RNA and DNA strands and separates the two strands.

5. Addition of Oligo d (G) primer :- Oligo d (G) primer is added to the reaction mixture and the temperature is maintained at 55 degree centrigrade. The oligo d (G) primer gets bound with oligo d (C) tail of the cDNA and mRNA by interchain hydrogen bonding. The primer provides 3'-OH group to proceed polymerization reaction.

6. Synthesis of the second DNA strand :- To the reaction mixture , klenow enzyme and all the four types of deoxyribonucleotides are added. The enzyme adds complementary nucleotides one by one to the 3'-OH group of the primer. As a result , a double stranded DNA called cDNA clone is formed from each cDNA.