Enzymes in rDNA or Recombinant DNA Technology

The major discovery that led to the birth of rDNA technology was the discovery of restriction enzymes or enzymes to cut DNA molecule.

Previously, we have discussed in detail the steps in rDNA technology. Let us start with definition.

What is recombinant DNA or rDNA technology?

It is the sum of techniques used in genetic engineering that involves the identification, isolation and insertion of gene of interest into a vector such as a plasmid or bacteriophage to form a recombinant DNA molecule and production of large quantities of that gene fragment or product encoded by that gene.

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Many enzymes are used in rDNA technology. Let us have a quick glance of the enzymes used in rDNA technology.

I. Nucleases: Enzymes to hydrolyse nucleic acids, DNA or RNA. Restriction Enzymes (called as Molecular scissors, Molecular scalpels etc)

a) Restriction endonuclease: Makes cuts at specific sites within the DNA Eg: Eco R1. Restriction site of Eco R1 is GAATTC.

b) Restriction exonuclease: Remove nucleotides from the ends.

c) Ribonuclease-H (RNase-H):

Selectively removes the mRNA from RNA DNA hybrid to separate the cDNA to synthesize the second strand.

It is isolated from retroviruses such as AMV, MMTV etc.

II. DNA Modifiers: Enzymes involved in modification of DNA strands to make it suitable for rDNA technology

a) DNA polymerase: It is involved in elongation of the strand. It is a template directed enzyme that adds complementary nucleotides to the free 3’ OH end of the strand. Commonly used DNA polymerase is Taq DNA polymerase isolated from Gram negative bacterium Thermus aquaticus

Uses: in Polymerase Chain reaction. In DNA sequencing by Maxam-Gilberst method. In making DNA probes for DNA finger printing.

b) Reverse Transcriptase: Enzyme that makes DNA strand using mRNA as template strand.

Also called as RNA dependant DNA synthetase. Used in the making of cDNA from mRNA for cDNA library preparation.

c) Alkaline Phosphatase :This enzyme removes terminal phosphate group (PO₄^-2 ) at the 5’ end of a DNA or RNA.

Uses: Removal of terminal phosphate group at the 5’ end of a DNA prevents self annealing of vector DNA .

In radioactive labeling using P32, phosphate group at the 5’ end is removed and replaced with P³² using alkaline phosphatase.

d) Polynucleotide kinase: It transfers or add phosphate from ATP to 5’OH group of dephosphorylated DNA or RNA.

Uses: To rephosphorylate the 5’ end of dephosphorylated DNA (vector DNA) or RNA.

Used in radiolabelling: to transfers radioactive P³² from ATP to dephosphorylated 5’ end of DNA or RNA.

e) Terminal nucleotidyl transferse : Enzyme that adds nucleotides to 3’OH group of a DNA fragment.

Uses: used to make homopolymer sticky or cohesive tails at 3’ end of DNA fragment. This helps in joining fragments with blunt ends.

Used to make radioactive DNA probes by end labeling.

f) Methyl transferase : Methyl Transferase: enzyme that adds a methyl group to cystine and adenine of DNA

Uses: To Methylate desired DNA in the rDNA to protect it from cleavage by restriction enzymes of restriction modification systems of the host. To protect restriction sites from a restriction enzyme , if the target DNA has many sites fro that particular restriction enzyme.

III.Ligases: ‘joining enzyme’

Joining enzyme that joins the ends of 2 double stranded DNA molecule. The process is called ligation.

The bond formed is called phosphodiester bond.

Between the 5’P of a nucleotide of one DNA fragment and the 3’ OH end of the other. Requires ATP and NAD⁺ for its activity. Eg: T4 ligase
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