What is a RESTRICTION

ENZYME?

 An enzyme
 Cleave DNA molecules at a specific
sequence of bases
 Nomenclature
Naming of a restriction enzyme :
 Genus (scientific) name
 Species name
 Strain Type
 Order
EcoRI E co R I
Genus Name :
Escherichia Strain : R
strain of
Species Escherichia
Name : Order : First
coli enzyme
coli
isolated from
the R strain.
 Restriction enzyme BamH I Hind III

Genus/scientific name of B : Bacillus H : Haemophilus

organism which isolated
the enzyme
Species name of organism am : amyloliquefaciens in : influenzae

Strain of the species H : H strain of Bacillus d : d strain of

amyloliquefaciens Haemophilus influenzae

Order of isolated enzyme First enzyme isolated from Third enzyme isolated
the H strain from the d strain
What if restriction enzyme does
not done their job precisely?
 What is Star Activity?
• The restriction enzyme cleaves the
restriction sequences that are SIMILAR but
not IDENTICAL to the defined sequence.
• It is only exibits under abnormal conditions.
Example:

C A A T T C
G T T A A C
• The recognition site of EcoRI under • The recognition site of EcoRI under
normal conditions abnormal conditions
Star activity under gel
electrophoresis Complete
digestion

Unwanted
digested
products
Pst1 Kpn1

both glycerol concentrations > 5%

• Replacing Mg2+ with Mn2+ • Raising the pH.
• The ionic strength. • If buffer contains too much
gylcerol or ethylene glycol.
 Incubation temperature

Most restriction enzymes show maximum activity at 37°C.

A few enzymes require higher or lower temperatures for optimal activity
e.g., Taq I, 65°C; Sma I, 25°C

*If the high temperature is incubated grater than 1 hour, the reaction can be
covered by a drop of mineral oil to prevent evaporation
 Presence of organic solvent
• Dimethyl sulfoxide (DMSO) :organosulfur compound with a high polarity and
high dielectric constant

• used in PCR to disrupt secondary structure formation in the DNA template.

• However, the effect of disrupted base-pairing imposed by DMSO introduces

the worthwhile consideration that mismatched base-pairing could result
during the primer annealing steps of PCR

• leading to increased mutation rates to the priming region.

Incubation temperature:
 Use the optimum temperature of respective restriction enzyme

Presence of organic solvent:

 Make sure the reaction is free of any organic solvents, such as
alcohols, which might be present in the DNA preparation.
• Restriction enzymes are stored in 50% gylcerol.
• Amount of enzyme added should not exceed 10% of the total reaction volume.
• Use standard 50uL reaction volume to reduce evaporation during incubation.
• Use only Mg2+ as the divalent cation in the reaction buffer.
• Increase the ionic strength of the reaction buffer to 50-150mM
• Keep the pH in the range of 7.2-8.5
THE END
Q&A Session!!!