Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites. Restriction Endonucleases are the enzymes that cut the DNA on interest from specific sites.

1. RESTRICTION ENZYMES
By
Muhammad Adnan Qadar

2. Also termed as Restriction Endonucleases OR
REases OR RE
Functions as Molecular Scissiors/DNA cutters
RESTRICTION/DIGESTION
The process of restricting OR breaking down the
DNA into pieces
RESTRICTION DIGEST
The product (Fragments of Broken DNA)
obtained from restriction
Methyl-Transferase/Methylase OR Mtase
An enzyme which attaches -CH3 group to host
DNA to protect it from REase digestion
TERMS TO KNOW

3. Also termed as Restriction Endonucleases OR
REases OR RE
Functions as Molecular Scissiors/DNA cutters
RESTRICTION/DIGESTION
The process of restricting OR breaking down the
DNA into pieces
RESTRICTION DIGEST
The product (Fragments of Broken DNA)
obtained from restriction
Methyl-Transferase/Methylase OR Mtase
An enzyme which attaches -CH3 group to host
DNA to protect it from REase digestion
TERMS TO KNOW

4. NOBLE PRIZE
Were
awarded

5. ACTION OF REases

6. REases can produce Sticky and Blunt
ends
REases can be Iso-schizo-mers
Only Type II restriction endonucleases are
the are mostly used
Methylated DNA cannot be digested by
Reases
Some REases also have dual nature (i.g.,
Reases
and Mtases)
CHARACTERISTICS OF REase

7. REases have specific recognition
sequence at which it cuts a DNA
molecule
For example, the RE called PvuI
(isolated from Proteus vulgaris) cuts
DNA only at the hexanucleotide
5´-CGATCG-3´
PvuII, cuts at a different
hexanucleotide in this case
5´-CAGCTG-3´
Many RE are Isoschizomers (Different
RE but same target sites)
REases cleaves Phosphodiestr bonds of
Nucleotides
SPECIFICITY OF REases
Frequency of Recognition
Sequence
It is calculated
mathematically i.e., a
tetranucleotide appears
every 44=256nt and
Hexanucleotide repeated
every 46=4096nt
Size of restricted DNA
fragment
Depends upon frequency nt
sequence appearance

8. • Four(four cutters), Five(five cutters), or even longer
nucleotide sequences cutting REases are also available
e.g., Sau3A (from Staphylococcus aureus strain 3A)
recognizes GATC and AlwNI cuts CAGNNNCTG
DEGENERATE RECOGNITION SEQUENCES
• Some REases cut DNA at any one of a family of related
sites, e.g., HinfI recognizes GANTC, so cuts at GAATC,
GATTC, GAGTC, and GACTC.

9. MECHANISM OF RESTRICTON
Blunt Ends or Flush End cut
Make a simple double-stranded cut in the middle
of the recognition sequence
HindII, PvuII and AluI are examples of blunt end
cutters

10. Reases Cut at exactly
the same position
Usually by two or four
nucleotides
As bp can stick
together again
So these are called
sticky ends
Sticky Ends or Cohesive Ends

11. REases with different recognition sequences
may produce the same sticky ends
BamHI (recognition sequence GGATCC) and
BglII (AGATCT) are examples—both
produce GATC sticky ends

13. Measure 2 µg of DNA in 16 µg of the sample
Add required REase (BglII) pure in the mixture
Maintain pH 7.4 with Tris-HCl (500mM)
Mg+2 (100mM) and Nacl (500mM) for proper
activity of RE
Add dithiothreitol (DTT) 10mM to stablize and
preventing inactivation of RE
PERFORMING DIGESTION IN LAB

14. Maintain 37°C for 1h
Then deactivate REase
by exposing it for short
time to 70°C or by
adding EDTA which
binds to Mg+2 of REase
and deactivates its
function