Reminder:

All molecular techniques are based

on the chemical “personality” (or chemical
properties) of the DNA molecule (or nucleic acids)
 Enzymes use in
Molecular Genetics
1. Restriction endonucleases/enzymes
2. Ligase
3. DNA polymerase
Molecular scissors which isolated from bacteria where they are used
as Bacterial defense against viruses

Molecular scalpels to cut DNA in a precise and predictable manner

Enzyme produced by bacteria that typically recognize specific 4-8

base pair sequences called restriction sites, and then cleave both
DNA strands at this site

A class of endo-nucleases that cleavage DNA after recognizing a

specific sequence

Members of the class of nucleases

 Nuclease

Breaking the phosphodiester bonds that link

adjacent
nucleotides in DNA and RNA molecules
 Endonuclease
Cleave nucleic acids at internal position
 Exonuclease
Progressively digest from the ends of the
nucleic acid
molecules
 Endonuclease
Type Characteristics

I  Have both restriction and modification activity

 Cut at sites 1000 nucleotides or more away from recognition site
 ATP is required
II  It has only restriction site activity
 Its cut is predictable and consistent manner at a site within or
adjacent to restriction site
 It require only magnesium ion as cofactor
III  Have both restriction and modification activity
 Cut at sites closed to recognition site

 ATP is required
 Restriction Enzymes
 There are already more than 1200 type II enzymes isolated
from prokaryotic organism
 They recognize more than 130 different nucleotide sequence
 They scan a DNA molecule, stopping only when it recognizes a
specific sequence of nucleotides that are composed of
symetrical, palindromic sequence

Palindromic sequence:
The sequence read forward on one DNA strand is
identical to the sequence read in the opposite direction
on the complementary strand

 To Avoid confusion, restriction endo-nucleases are named

according to the following nomenclature
 Nomenclature
 The first letter is the initial letter of the genus name of
the organism from which the enzyme is isolated
 The second and third letters are usually the initial
letters of the
organisms species name. It is written in italic
 A fourth letter, if any, indicates a particular strain
organism
 Originally, roman numerals were meant to indicate the
order in which enzymes, isolated from the same
organisms and strain, are eluted from a
chromatography column. More often, the roman
numerals indicate the order of discovery
Nomenclature
Specificity
G G A T C C
C C C A G G
G G A T C C
C C C A G G

G A T C C
G
G
C C A G G
Restriction Product
 Restriction enzymes

Restriction enzymes can be grouped by:

 number of nucleotides recognized (4, 6,8 base-cutters most
common)
 kind of ends produced (5’ or 3’ overhang
(cohesive=sticky), blunt=flush)
 degenerate or specific sequences
 whether cleavage occurs within the recognition
sequence
A restriction enzyme (EcoRI)
1. 6-base cutter
2. Specific palindromic
sequence
(5’GAATTC)
3. Cuts within the
recognition
sequence (type II
enzyme)
4. produces a 5’
overhang (sticky end)
 Application
 Genetic engineering