Topic Isolating, Cloning and Sequencing DNA: Q) Name all the enzymes involved in the large-scale production of a named important eukaryotic protein by the genetically engineered E.coli cells. State their functions.

Ans :

Making of the double-stranded cDNA (in the test tube)

1) Reverse transcriptase – to make cDNA from mRNA template forming DNA : RNA hybrid

2) RNAse – to digest the RNA template (can also use heat by boiling or alkaline hydrolysis w/o enz)

3) DNA polymerase (Klenow fragment) – to make 2^nd strand cDNA

Making of the recombinant vector (in the test tube)

4) Restriction enyzme / endonuclease – to digest gene of interest and plasmid vector

5) DNA ligase – to seal nick b/w gene and vector, to form recombinant vector

Replication of the gene (in the bacteria)

6) Helicase – to unwind double stranded plasmid at its origin of replication

7) Primase – to synthesize RNA primer DNA polymerase – to replicate plasmid DNA (there are actually two types of DNA polymerase I and III involved) 

8) Topoisomerase – to relieve strain of double stranded DNA ahead of the replication fork due to the unwinding of the DNA double helix by helicase

9) DNA ligase – to join the 2 okazaki fragments of the lagging strand

Expression of the gene (in the bacteria)

10) RNA polymerase – to synthesize mRNA of gene

11) Aminoacyl-tRNA synthetase – to attach aa to its specific tRNA

12) Peptidyl-transferase (of ribosomal large subunit) – to form peptide bonds b/w aa

Comment :

Some of the enzymes are not that important for the A level exams (e.g. Klenow fragment, topoisomerase) but some schools may teach or briefly outline during lesson