Question

What are restriction enzymes, and why are they used in recombinant DNA technology?

Answer 1

Restriction enzymes are used to cut the DNA sequences at specific sites in sequences which are recognized by individual enzymes. They are extracted from bacterial species by isolation of these enzymes occurring naturally in the bacterial species which provide them a defense against viruses and bacteriophages. They are isolated and named by the bacterial species (first letter representing the genus and the next two representing the species), their strain and the order of their discovery. For example HindIII is named after Haemophilus influenzae strain Rd and it was discovered 3rd in the bacteria after HindI and HindII.

H - Haemophilus

in - influenzae

d - strain Rd

III- order in which discovered

They require co factors to cleave the DNA molecule. Based on the site of cleavage and co factors they are divided into 4 types. Enzymes that require SAM (S-adenosyl-L-methionine) and ATP and cleave the DNA at about 1000bp away from the recognition site are called Type I restriction enzymes (example: EcoKI) and those that require Magnesium as their co factor and cleave at short distances away from the recognition site are called Type II restriction enzymes (example: BamHI). Type III restriction enzymes also cleave at short distances but they require ATP and SAM (example: HinfIII). Type IV enzymes can recognize DNA sequences that are modified by methylation etc., and they have a low sequence specificity (example SauUSI). Restriction Enzymes cut the DNA into fragments by cleaning their sugar phosphate backbone between two nucleotides. Depending on their cutting site, restriction enzymes can produce two types of ends blunt and sticky ends.

For example EcoRI recognizes the sequence GAATTC and cleaves at the site between G and A forming sticky ends that are free nucleotides

5'G AATTC3'

3'CTTAA   G5'

while enzymes like HaeIII recognize the sequence GGCC and cut between the 2nd guanine base and the cytosine base forming blunt ends which are not free nucleotides as they are base paired with their complementary nucleotide.

5'GG CC3'

3'CC GG5'

Restriction enzymes are of great use in the field of biotechnology. They are used for restriction digestion of PCR products to identify the SNPs on a gene sequence. They are also used in blotting experiments by using them to digest the DNA sequences. They are majorly used in rDNA technology experiments like cloning where they are used to cut a specific gene sequence and insert it into the plasmid (which is also cut by a restriction enzyme) and they are ligated. The plasmid acts as a vector and is transformed into the host bacteria which incorporates the gene into it's genome which are then allowed to multiply and form colonies.