In this lucid slide show we will go through the very basic tools of biotechnology: Restriction Enzymes and qualities of a good Plasmid Vector.

1. Somnath Mondal
Introduction To Plant Biotechnology
Part I

2. Somnath Mondal
Gene of interest present in the
DNA of Plant A
To be incorporated into Plant B
 Gene of interest copied by PCR.
 The gene sequence can be
synthesized by oligonucleotide
synthesizer.
 Gene is incorporated into
plasmid vector DNA.
 Plasmid vector with gene of
interest in incorporated into
bacteria, like E.coli.
 Plasmid vector with gene of
interest is isolated from E.coli.
 Plasmid vector with gene of
interest is isolated from E.coli is
again incorporated into
Agrobacterium tumefaciens.
SELECTION
SELECTION
SELECTION

3. Somnath Mondal
Basic tools of RDT
Restriction Enzyme Plasmid DNA

4. Somnath Mondal
Restriction Enzymes
 Werner Arber, Hamilton Smith and Daniel Nathans got Nobel Prize in Physiology orMedicine in 1978 for
their research on Restriction Endonucleases.
 They cut the phosphodiester bond that joins adjacent nucleotides in a DNA strand and thus make internal
cuts in DNA.
 Within bacteria, they provide protection against virus infection as they cleave the viral nucleic acid and
nullify their pathogenicity.

5. Somnath Mondal
 Restriction enzymes recognize palindromic DNA sequences – which read same forward and backward on
opposite strands of DNA.
 MADAM, REVIVER, REFER, LEVEL, CIVIC, RADAR – are some common PALINDROMIC WORDS which read the
same forward or backward.
 Some common Palindromic DNA Sequences are:
5'…..GAATTC…..3' 5'…..GGATCC…..3'
3'…..CTTAAG…..5' 3'…..CCTAGG…..5'

6. Somnath Mondal
Types of Restriction Endonuclease
Restriction
Endonuclease
Type I Type II Type III Type IV Type V
• Cuts at sites away
from recognition site.
• Multifunctional
protein with both
restriction
endonuclease and
methylase activity.
• Cuts within
recognition site or
in short distance
from it.
• Unifunctional
protein with only
restriction
endonuclease
activity.
• Cuts at sites in
short distance
from recognition
site.
• Multifunctional
protein with both
restriction
endonuclease and
modification
methylase
activity.
• They target
modified DNA
like methylated
DNA.
• They use guide
RNAs to identify
specific non-
palindromic
sequences and
cut the DNA.

7. Somnath Mondal
Nomenclature of
Restriction Endonucleases
1st Letter of the enzyme = 1st Letter of the Genus of the bacterium where from it is isolated.
2nd & 3rd Letter of the enzyme= First two letters of the species of the bacterium where from it is isolated.
4th Letter= Serotype or strain of the bacterium where from it is isolated.
Number= Numbering of the enzyme isolated from the same bacterium according to its order of discovery.
e.g. EcoR1= Isolated from Escherichia coli,
Strain: RY13
1st Isolated Enzyme from that bacterium.

8. Somnath Mondal
Sticky End & Blunt End Cutters
5'
C C C G G G
G G G C C C
5'
5'3‘
3'
C C C G G G
G G G C C C
5'
5'3‘
3'
5'
3‘
3‘
5'
3'C C C G G G
G G G C C C
5'
3‘
C T T A A G
G A A T T C5'
5'
3'
3'
C T T A A G
C T T A A
G A A T T C5'
3' 5'
3'
5'
G A A T T C
5'
3'
3' 3'
EcoRI SmaI
5'
3'
Sticky Ends
Blunt Ends

9. Somnath Mondal
• Plasmid DNA is a small circular extrachromosomal DNA
found in bacteria.
• They are approximately 1 to 4 kb in size.
• They can replicate independent of bacterial chromosome.
• They can be used as Vectors to carry gene of interest and
also to multiply their copy.
Plasmid Vectors

10. Somnath Mondal
Important Features of DNA Cloning Vectors
– Origin of replication (ori) – site for DNA replication that allow plasmids to
replicate independently from host chromosome.
– Multiple cloning site (MCS) – recognition sites for several restriction
enzymes in which gene of interest can be inserted.
– Selectable marker genes – allow to select for transformed colonies.
– RNA polymerase promoter sequences – used for transcription in vitro and
in vivo.
– DNA sequencing primers.

11. Somnath Mondal
EcoRI
HindIII
PstI
BamHI
NcoI
SpeI KpnI
SalI
BstXI
SacII
SspI
BlpI
Ori
R
P
P
BSM
Bacterial Selection Marker
P
S
M
PlantSelectionMarker
....GAATTC….
…CTTAAG….
A vector map:

12. Somnath Mondal
Ori
Importance of Ori site:

13. Somnath Mondal
GAATTC GGATCC
CTTAAG CCTAGG
OR
OR
EcoRI
EcoRI
BamHI
BamHI
EcoRI BamHI
Ligation
Plasmid Vector
Plasmid Vector
Gene of interest
Transfer of GI into vector:

14. Somnath Mondal
Bacterial Transformation & possibilities:

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lacZ
Bacteria with no plasmid Bacteria with
only plasmid
Bacteria with
plasmid with
gene of interest
PETRI-PLATES WITH LURIA BERTANI AGAR (LBA) MEDIUM WITH KANAMYCIN ANTIBIOTIC AND X-gal
They can’t grow on a plate
containing kanamycin antibiotic.
They can grow on a plate
containing kanamycin antibiotic
and the colour of colony will be
blue.
They can grow on a plate
containing kanamycin antibiotic
and the colour of colony will be
white.
Selection:

16. • Presence of active lacZ gene will produce β-galactosidase enzyme.
• This enzyme cleaves the X-gal into 5-bromo-4-chloro-indoxyl.
• Dimerization & oxidation of this compound form a blue coloured compound, 5,5‘-dibromo-4,4‘-
dichloro-indigo.
lacZ
Reason behind blue-white screening:
Somnath Mondal

17. Somnath Mondal
So, whats your choice? Blue or white?