Restriction Enzyme Presentation

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

Genus Name : Escherichia Species Name : coli

co

R

Strain : R strain of Escherichia coli

I

Order : First enzyme isolated from the R strain.

Restriction enzyme Genus/scientific name of organism which isolated the enzyme

BamH I B : Bacillus

Hind III H : Haemophilus

Species name of organism am : amyloliquefaciens

in : influenzae

Strain of the species

H : H strain of Bacillus amyloliquefaciens

d : d strain of 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 normal conditions

• The recognition site of EcoRI under abnormal conditions

Star activity under gel electrophoresis Complete digestion

Unwanted digested products

Pst1

Kpn1

both glycerol concentrations > 5%

• Replacing Mg2+ with Mn2+ • The ionic strength.

• Raising the pH. • 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

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