VPB 321: Animal Biotechnology (2+1)

RESTRICTION ENZYMES

• A Restriction Enzyme (or restriction endonuclease) is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Restriction enzymes are found in bacteria.
• To cut the DNA, a restriction enzyme makes two incisions, once through each sugar-phosphate backbone (i.e. each strand) of the DNA double helix.
• Recognition sequences vary between 4 and 8 nucleotides, many of them are palindromic
• Recognition sequences in DNA differ for each restriction enzyme, producing differences in the length, sequence and strand orientation
• There are four classes of restriction endonucleases: types I, II, III and IV. All types of enzymes recognize specific short DNA sequences and carry out the endonucleolytic cleavage of DNA to give specific double-stranded fragments with terminal 5'-phosphates.
• Type II are the most commonly available and used restriction enzymes.
• Each enzyme is named after the bacterium from which it was isolated using a naming system based on bacterial genus, species and strain.
• For example, EcoRI
• E – Escherichia –Genus
• Co – coli –species
• R-strain
• I-order of identification

Applications of Restriction enzymes

• Restriction enzymes are used to assist insertion of genes into plasmid vectors during gene cloning and protein expression experiments.
• Restriction enzymes are used to digest genomic DNA for gene analysis by Southern blot
• Restriction enzymes can also be used to distinguish gene alleles by specifically recognizing single base changes in DNA known as single nucleotide polymorphisms (SNPs).