Download Restriction Enzymes

Restriction Enzymes
• Originally discovered by scientists
when studying bacteria’s defense
mechanisms toward bacteriophages
(viruses)
• Named after the organism that it was
discovered from and the number of the
order it was discovered
• Ecor I…Escherichia coli 1st discovered
• HindI II…Haemophilus influenza 2nd discovered
Restriction Cuts
• Recognizing cuts…sticky vs blunt
• EcorI…cuts between G&A
• 5’-GAATTC-3’
• 3’-CTTAAG-5’
• Creates a 5’ overhang
• PstI…cuts between G&A
• 5’-CTGCAG-3’
• 3’-GACGTC-5’
• Creates a 3’ overhang
• PvuII…cuts between C&G
• 5’-CAGCTG-3’
• 3’-GTCGAC-5‘
• Creates a blunt end
Evaluating the cuts of a Restriction Enzyme
• Palindromes in DNA occur
randomly throughout the
genome which allows for
multiple cuts with a single
restriction enzyme. Calculating
# of cuts
• 4 bp enzyme occurs ~300bp
• 6 bb enzyme cut ~3000bp
• Lambda genome is ~48500bp
while its plasmid is ~6000bp
Using Restriction Enzymes to Clone Genes of
Interest
• Since the amount of cuts in the
standard genome is too many for
controlling gene insertion and
regulation, plasmid cloning vectors
are preferred
• Restriction cuts are often singular
• The cuts can be selected to be
upstream from a promotor region to
ensure expression
• Insertion vectors are engineered to
have the complementary sticky ends
• Ends are tied with the enzyme ligase
Restriction Activity & Enzyme Strength
• Enzymes are measured in units of
activity (U)
• U = to amount necessary to digest 1ml of
lambda DNA
• Most are supplied at 10 to 20U/ml
• Operating temperature for most is 37C
• Peak effective time is 1hr
• Enzymes (like most DNA) are temperature
sensitive
• Most stored in glycerol at -20C
• Glycerol will not solidify creating a
super cold liquid
• Should ONLY be removed from cold long
enough to handle