Restriction Enzymes
•Restriction enzymesare part of the defense system of bacteria: they digest foreign DNA
that enters the bacterial cell.
•Each species of bacteriahas its own set of restriction enzymes. Each enzyme cuts DNA at
a specific short base sequence.For instance, EcoR1 cuts the DNA at the sequence GAATTC,
and BamH1 cuts at GGATCC. There are hundreds of restriction enzymes known.
•Using properly chosen enzymes, the gene you want can be cut out of the chromosome
intact, with very little extra DNA.
Many restriction enzymes give a staggered cut across the DNA double helix.This produces
short single stranded regions, called “sticky ends”.The ends are sticky because they
spontaneously pair with similar ends.
Restriction Endonucleases􀁺
Restriction endonucleases cut DNA􀁺
Present in bacteria􀁺
Cut at sequence specific sites–Usually 4 or 6base pairs long􀁺
Bacteria use them to destroy ‘foreign’DNA–Bacteria protect their own DNA against selfcutting by special methylationof their DNA􀁺
Restriction enzymes can be purified and are used in genetic engineering studies
Example Restriction enzymes
–EcoR I (E. coliRestriction Endonuclease I)
–StuI (StreptomycestubercidicusI)
Molecular Scissors and Glue􀁺
There are 200’s of restrictionenzymes, each one with a different recognition site–These
enzymes are ‘molecular scissors’and can be used to specifically cut long DNA strands
into smaller pieces
called "restriction enzymescalled enzymes““because restrict host range for certain
bacteriophagebacteriophage􀁺
􀁺bacterial" immune system": destroy any "nonbacterial" non--self" DNAself" DNA􀁺
methylaserecognizes same sequence in host DNA and protects it by methylatingit;
restriction enzyme destroys unprotected = non-self DNA (restriction/modification
systems
Characteristic Restriction Enzymes
􀁺Most recognize and cut palindromicsequences
􀁺Many leave staggered (sticky) ends
􀁺by choosing correct enzymes can cut DNA very precisely
􀁺Important for molecular biologists because restriction enzymes create
unpaired "sticky ends" which anneal with any complementary sequence
􀁺Hundreds of restriction enzymes have been identified.
The activity of restriction enzymes is dependent upon precise environmental
condtions:
PH
Temperature
Salt Concentration
Ions
An Enzymatic Unit(u) is defined as the amount of enzyme required to digest 1 ugof
DNA under optimal conditions
3-5 u/ugof genomic DNA
1 u/ugof plasmid DNA
Stocks typically at 10 u/ul