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1. By:
Arpita Mukherjee

3. DNA RECOMBINE DNADNA CELLS
CELLS : MICROORGANISM/PLANT CELLS/ANIMAL CELLS etc.
Isolate the DNA
Restriction
endonuclease
Gene of interest
vector

4. Restriction
Endonuclease
• Types: Class I, class II and
class III
• Palindromic sequences
• Recognition sites
• Cleavage Breaking the
Phosphodiester bond
• Ends produces Either
Blunt
or Sticky
Nomenclature:
EcoRI:
E: Escherichia
co: coli
RI: strain
HindIII:
H: Haemophilus
in: influenza
dIII: strain
TYPE I: Modification & restriction
activity, 1000bp away
TYPE II: Single separate proteins for
restriction & modification, cut at
same site
TYPE III: Modification & restriction
activity are separate 25bp away
EcoRI: GAATTC G AATTC
CTTAAG  CTTAA G
EcoRV: GATATC GAT ATC
CTATAG  CTA TAG

5. CELL1 CELL2
ISOLATED DNA
TREATED WITH RESTICTION ENDONUCLEASE
A. Both the cells with same enzyme: Blunt end or Sticky end
B. Both the cells with different enzyme:
Cell 1: Blunt end
Cell 2: Sticky end
Cell 1: Sticky end
Cell 2: Blunt end
Cell 1: Sticky end Non Compatible
Cell 2: Sticky end
 Modifications of cut ends:
linker
adaptor
homopolymer tailing
trimming

6. DNA RECOMBINE DNADNA CELLS
CELLS : MICROORGANISM/PLANT CELLS/ANIMAL CELLS etc.
Isolate the DNA
Restriction
endonuclease
Artificial gene synthesis/ cDNA synthesis/ RTase Method

7. Gene of Interest isolated from cell Compatible vector
LIGASE
TRANSFORMATION

8. By:
Arpita Mukherjee