Bio tech class 12th

1. © 2021, BYJU'S. All rights reserved
© 2021, BYJU'S. All rights reserved
Biotechnology : Principles and Processes
Notes
Classes
Plasmids as Vectors, Nucleases, Restriction Endonucleases and
Insertion of GOI Plasmid

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Plasmids as Vectors
Nucleases
Summary
1
2
Key Takeaways
Restriction
endonucleases
Insertion of GOI into
Plasmid
3
4

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Recall! Our Goal
rDNA technology
Technology used for introducing DNA sequence from one organism to another
Our goal is to produce our desired protein human insulin from E.coli
using recombinant DNA technology in large quantity.

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Recall! The Roadmap
1
2
3
4
Identification of
Gene of interest
(GOI)
Isolation
of DNA
Amplification
of GOI
Separation
and elution
of GOI
5
Modification of
GOI by PCR
Gene of
interest
(GOI) Insulin
GOI
PCR
Purified
DNA pellet GOI Modified GOI
PCR
Amplified GOI
Treated cell extract
in chilled ethanol

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Recall! The Vectors
DNA fragment
(Gene of
interest)
BAC
(Vector)
Recombinant
DNA
Vectors act as the carriers or vehicle for the DNA fragment
while transferring it into the host cell.

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Recall! Bacteriophage and its Life Cycle
Bacteriophage
Bacterial genome
Bacteria
Bacteriophage
Bacteriophage DNA
Life cycle of bacteriophage inside bacteria
● Bacteriophages or phages are the viruses that infect bacterial cells and grow on them.
● Many bacteria are susceptible to these viruses.
● These viruses attach to the surface of bacterial cells, inject their genetic material, and use the
bacterial enzymes to multiply while destroying the bacterial cells.

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● After the isolation of GOI, it needs to be inserted into the host (here E. coli) to
produce the desired protein (Insulin) in the host.
● However, the GOI cannot be inserted directly into the host. Hence, it requires a
vector to take it into the host.
Plasmids as Vectors
● Plasmids are the most commonly used
vectors.
● Plasmids are autonomously replicating
extrachromosomal circular DNA found in
bacteria.
● To insert the GOI, the circular plasmid
needs to be cut open which can be done
by endonucleases.
Plasmid

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Nucleases are the enzymes that cleave the nucleic acids i.e. DNA and RNA by
breaking down the phosphodiester bonds.
Nucleases
Nucleases
Exonucleases
Endonucleases
Make cuts within the
DNA molecule
Make cuts at the ends
of the DNA molecule
Based on region of action
5’
3’
3’
5’
5’
3’
3’
5’
5’
3’
3’
5’
3’
5’
5’
3’
5’
3’
3’
5’

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● Restriction endonucleases are a type of endonucleases found in certain bacteria that
cut the DNA molecule at or near specific nucleotide sequences.
Discovery
Restriction Endonucleases
● It was discovered as a part of
restriction modification system in
bacteria which is used by bacteria to
protect its own genome and destroy
the foreign DNA (e.g. bacteriophage
DNA).
Restriction modification
system
Restriction enzyme Modification enzyme

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Restriction Endonucleases
Restriction
modification
system
Restriction
enzyme
Modification
enzyme
It recognizes and cuts up the foreign
bacteriophage DNA.
It recognizes and modifies the bacterial
DNA by adding methyl groups to
protect it from the DNA-degrading
activity of its own restriction enzyme.
Bacterial genome
Methyl group
Bacteria
Cleaved bacteriophage
DNA
Bacteriophage
Bacteriophage DNA
Discovery

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Escherichia coli
Restriction modification system was
discovered in E. coli.
Did You Know?

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Restriction Endonucleases
Mode of action
● Restriction endonucleases scan the DNA for certain specific sequences of
nucleotides.
● The specific sequences which are recognized by the restriction enzymes are called
recognition sequences or recognition sites.
● Recognition sites help the restriction endonucleases to identify the cut site in the
DNA.
A G T T A G T C A
T C A A T C A G T
T C A A T C A G T
A G T T A G T C A
5’
3’
5’
3’
C C C
G G G
G G G
C C C
5’ CCCGGG 3’
3’ GGGCCC 5’
SmaI
Recognition sequence of SmaI

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Did You Know?
● More than 900 restriction enzymes
have been isolated from over 230
strains of bacteria.

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Restriction Endonucleases
Palindromic sequences
● In English, palindromes refers to the words which read the same from left to right and
vice versa. E.g. MALAYALAM, LEVEL, ROTATOR etc.
● Some restriction enzymes have recognition sequences that reads the same on both
the strands when the orientation of reading is kept the same, i.e. 5’ 🡪 3’.
● These sequences of base pairs in the DNA are known as palindromic sequences.
● Eg: EcoRI cuts the DNA between bases G and A only when the sequence GAATTC is
present in the DNA.
M A L A Y A L A M
Forward
Backward
T
A
5’
3’ 5’
3’
G
A C T
T
T C G A A

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Restriction Endonucleases
Action of restriction enzymes
● Restriction enzymes cut DNA into fragments of a size suitable for cloning.
● The restriction enzymes produce 2 types of cuts in DNA.
Restriction enzymes
Sticky
Blunt
Based on the type of cut
5’
3’
3’
5’
Recognition sequence
G G G
C C C
C C C
G G G
5’
3’
3’
5’
G G G
C C C
C C C
G G G
3’
5’
5’
3’
5’
3’
3’
5’
Recognition sequence
A A T T C
G
G
C T T A A
5’
3’
3’
5’
A A T T C
G
G
C T T A A
3’
5’
5’
3’
Overhang
EcoRI
SmaI

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Restriction Endonucleases
Restriction enzymes and types of cuts
Restriction Enzyme Recognition site Type of cut
BamHI Sticky
EcorI Sticky
HindIII Sticky
PstI Sticky
PvuII Blunt
SmaI Blunt
5’- CAGCTG -3’
3’- GTCGAC -5’
5’- CCCGGG -3’
3’- GGGCCC -5’
5’- GAATTC -3’
3’- CTTAAG -5’
5’- GGATCC -3’
3’- CCTAGG -5’
5’- AAGCTT -3’
3’- TTCGAA -5’
3’- CTGCAG -3’
5’- GACGTC -5’

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Restriction Endonucleases
Nomenclature
● Restriction endonucleases are named according to the organism in which they were
discovered, using a system of letters and numbers.
● The nomenclature follows certain rules.
3
1 2 4
2nd
Part
First 2 letters of the
species name
4th
Part
Order of discovery in
Roman numerals
1st
Part
First letter of the
genus name
3rd
Part
First letter of
Strain type
● Example: EcoRI (isolated from Escherichia coli RY 13 strain)
R
E co I
2nd
Part
First 2 letters of the
species name
Escherichia coli
4th
Part
Order of discovery in Roman numerals
First (I) Restriction enzyme to be
discovered in E. coli
1st
Part
First letter of the
genus name
Escherichia coli
3rd
Part
Strain type
RY strain

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Insertion of GOI into Plasmid
3’
5’ A A T T C
G
3’
5’
A A T T C
G
G
C T T A A
G
C T T A A
Requirements
Plasmid DNA with the restriction site
EcoRI recognition site
EcoRI
5’
3’
3’
5’
A A T T C
G
G
C T T A A
Gene of interest (GOI) with flanking sequences (after PCR)
Suitable Restriction endonuclease Ligase
(flanking sequences having the suitable restriction site)
● A suitable plasmid vector is required to carry
the GOI into the host.
● A plasmid has several restriction sites which
can be exploited to insert the GOI.
● The GOI is required to have suitable
restriction sites in its flanking sequences
(The same restriction sites should be
present in the plasmid).
● A suitable restriction enzyme is required
which can produce the same sticky ends in
plasmid and GOI.
● Ligase is required to make the
phosphodiester bonds between the DNA
fragments to join plasmid and GOI together.

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Insertion of GOI into Plasmid
Generation of sticky ends in plasmid and GOI
EcoRI recognition site
5’
3’
3’
5’
A A T T C
G
G
C T T A A 3’
5’ A A T T C
G
3’
5’
A A T T C
G
G
C T T A A
G
C T T A A
EcoRI EcoRI EcoRI
5’
3’
3’
5’
A A T T C
G
G
C T T A A 3’
5’ A A T T C
G
3’
5’
A A T T C
G
G
C T T A A
G
C T T A A
Plasmid with sticky ends
3’ 5’
A A T T C
G
G
C T T A A
5’ 3’
GOI with sticky ends
EcoRI recognition site EcoRI recognition site
Digestion with suitable restriction endonuclease Digestion with same restriction endonuclease

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Insertion of GOI into Plasmid
Complementary pairing of sticky ends of Plasmid with GOI
5’
3’
3’
5’
A A T T C
G
G
C T T A A
Plasmid with
sticky ends
3’ 5’
A A T T C
G
G
C T T A A
5’ 3’
GOI with sticky ends
3’
5’
5’
3’
5’
3’
3’
5’
A A T T C
G
G
C T T A A
A A T T C
G
G
C T T A A
Gaps in the sugar
phosphate backbone
Complementary base pairing of sticky ends
● The sticky ends of the plasmid and gene of interest (GOI) stick together by
complementary base pairing.
● But after complementary base pairing, the junction of plasmid DNA and GOI DNA has
gaps in the sugar-phosphate backbone due to the lack of phosphodiester bonds.

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Insertion of GOI into Plasmid
5’
3’
3’
5’
Gap filling with ligase
A A T T C
G
G
C T T A A
A A T T C
G
G
C T T A A
Gaps in the sugar
phosphate backbone
Digestion with ligase
5’
3’
3’
5’
A A T T C
G
G
C T T A A
A A T T C
G
G
C T T A A
Ligase Ligase
Ligase Ligase
5’
3’
3’
5’
A A T T C
G
A A T T C
G
G
C T T A A
G
C T T A A
Plasmid with GOI
(Recombinant DNA)
● Ligase enzyme links the 5’
phosphate and 3’ OH of
two fragments by making a
phosphodiester bond
between them (process
named ligation).
● After ligation, the process
of insertion of GOI into the
plasmid completes and
hence a recombinant DNA
is formed.
● In the next step, the
recombinant plasmid DNA
is inserted into the host.
E. coli

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Summary
● Plasmids
○ Plasmids are autonomously replicating extrachromosomal circular DNA found in
bacteria.
○ To insert the GOI, the circular plasmid needs to be cut open which can be done by
endonucleases.
● Nucleases
○ Nucleases are the enzymes that cleave the nucleic acids i.e. DNA and RNA by breaking
down the phosphodiester bonds.
● Restriction endonucleases
○ Restriction endonucleases are a type of endonucleases found in certain bacteria that cut
the DNA molecule at or near specific nucleotide sequences.
Restriction enzymes
Sticky
Blunt
Based on the type of cut

23. © 2021, BYJU'S. All rights reserved
Summary
Nomenclature of restriction endonucleases
● Restriction endonucleases are named according to the organism in which they were
discovered, using a system of letters and numbers.
● The nomenclature follows certain rules.
3
1 2 4
2nd
Part
First 2 letters of the
species name
4th
Part
Order of discovery in
Roman numerals
1st
Part
First letter of the
genus name
3rd
Part
Strain type

24. © 2021, BYJU'S. All rights reserved
Summary
EcoRI recognition site
5’
3’
3’
5’
A A T T C
G
G
C T T A A 3’
5’ A A T T C
G
3’
5’
A A T T C
G
G
C T T A A
G
C T T A A
EcoRI EcoRI EcoRI
5’
3’
3’
5’
A A T T C
G
G
C T T A A
Plasmid with sticky ends
3’ 5’
A A T T C
G
G
C T T A A
5’ 3’
GOI with sticky ends
EcoRI recognition site EcoRI recognition site
5’
3’
3’
5’
A A T T C
G
G
C T T A A
A A T T C
G
G
C T T A A
Complementary base pairing of sticky ends
5’
3’
3’
5’
A A T T C
G
G
C T T A A
A A T T C
G
G
C T T A A
Ligase Ligase
Ligase Ligase
Gap Gap
Gap
Gap
5’
3’
3’
5’
A A T T C
G
A A T T C
G
G
C T T A A
G
C T T A A
Plasmid with GOI (Recombinant DNA)
E. coli
Insertion of GOI into plasmid