BIOTECH 2

 

RESTRICTION ENZYMES

• Restriction enzymes belong to a larger class of enzymes called
• Restriction enzymes are called as molecular scissors because these enzymes cut DNA at specific sites.
• The first restriction endonuclease is Hind II.
• The restriction enzymes cut DNA at specific base sequence, and these specific base sequence is known as the recognition sequence.
• The convention for naming restriction enzymes –
• The first letter of the name comes from the genus.
• The second two letters come from the species of the prokaryotic cell from which they were isolated, e.g., EcoRI comes from Escherichia coli RY 13.
• In EcoRI, the letter ‘R’ is derived from the name of strain.
• Roman numbers following the names indicate the order in which the enzymes were isolated from that strain of bacteria.
• 900 restriction enzymes that have been isolated from over 230 strains of bacteria.

These are of two kinds

1. Exonucleases

2. Endonucleases

• Exonucleases remove nucleotides from the ends of the DNA whereas, endonucleases make cuts at specific positions within the DNA.
• Each restriction endonuclease recognizes a specific palindromic nucleotide sequences in the DNA.
• The palindrome in DNA is a sequence of base pairs that reads same on the two strands when orientation of reading is kept the same.

Example- the following sequences reads the same on the two strands in 5' à 3' direction, this is also true if read in the 3' à 5' direction.

 5' —— GAATTC —— 3’

 3' —— CTTAAG —— 5'

• Restriction enzymes cut the strand of DNA a little away from the center of the palindrome sites, but between the same two bases on the opposite strands which leaves a single stranded portions at the ends and the overhanging stretches called sticky ends on each strand.
• When cut by the same restriction enzyme, the resultant DNA fragments have the same kind of ‘sticky-ends’ and, these can be joined together using DNA ligases.