*
M
E STUDY OF CLONING VECTORS, RESTRICTION ENDONUCLEASES AND DNA LIGASE
R
G
E
F PRIOR CONCEPT
O
R WHAT ARE VECTORS?
A vector (Vehicle) is a DNA molecule which is used for transporting exogenous DNA into the host
M cell.
A WHAT IS CLONING?
T Cloning in biotechnology refers to the process of creating clones of organisms or copies of cells or
DNA fragments.
2
❖ CLONING VECTOR
A cloning vector is self-replicating DNA molecules into which a desired DNA fragment is
inserted. They carry desired DNA into the host cell and replicate itself and desired DNA into
the host.
Cloning vector facilitates amplification of a single copy DNA molecule into many copies.
Molecular gene cloning is difficult without the use of the cloning vectors.
The cloning vectors must possess the following general characteristics:
⮚ It should small in size.
⮚ It should be easily introduced into the
host cell.
⮚ It should have the ability to replicate
autonomously in the host cell.
⮚ It must also be compatible with the
host organism.
⮚ The vector should have marker site
that allow easy detection of transformed cell.
⮚ It must possess a restriction site.
TYPES OF CLONING VECTOR
o Plasmids
o Bacteriophages
o Cosmids
o Artificial chromosomes
o Plasmids
Extra-chromosomal genetic material found in the cytoplasm of bacterial cell is known as
plasmid.
P Plasmid carries genes for antibiotic resistance but do not have any important functions.
A
G
E
Notes by S.D Sir
, *
M
E STUDY OF CLONING VECTORS, RESTRICTION ENDONUCLEASES AND DNA LIGASE
R
G
E ✔ Discovered by Hays & Lederberg.
F
O ✔ Plasmid are circular, double stranded and spiral molecules.
R
M ✔ Plasmid are capable of Independent
A Replication.
T
✔ Plasmid are not essential for the growth and
2 survival of bacteria.
✔ Plasmid has restriction site and marker gene
(antibiotic resistance gene).
Feature that are required to facilitate cloning:
1. ORI → Origin of replication. This
makes autonomous replication in vector.
2. Cloning Site (Restriction site) →
Cloning site is a place where we can
make a cut by restriction site and foreign
DNA can be added.
3. Selectable marker → A gene that
helps to select the transformed cell with
desired gene from non-transformed cell.
So, gene is marker gene.
▪ Nomenclature of plasmid cloning vector: pBR322 cloning vector has the following
elements:
● p= Plasmid
● B= Bolivar (name of the scientist)
● R= Rodriguez (name of the scientist)
● 322= Number given to distinguish
this from other developed from
same laboratory.
“First genetically engineered plasmid
widely used in rDNA technology”
It has;
P i. ORI (Origin of replication).
A
G
E
Notes by S.D Sir
M
E STUDY OF CLONING VECTORS, RESTRICTION ENDONUCLEASES AND DNA LIGASE
R
G
E
F PRIOR CONCEPT
O
R WHAT ARE VECTORS?
A vector (Vehicle) is a DNA molecule which is used for transporting exogenous DNA into the host
M cell.
A WHAT IS CLONING?
T Cloning in biotechnology refers to the process of creating clones of organisms or copies of cells or
DNA fragments.
2
❖ CLONING VECTOR
A cloning vector is self-replicating DNA molecules into which a desired DNA fragment is
inserted. They carry desired DNA into the host cell and replicate itself and desired DNA into
the host.
Cloning vector facilitates amplification of a single copy DNA molecule into many copies.
Molecular gene cloning is difficult without the use of the cloning vectors.
The cloning vectors must possess the following general characteristics:
⮚ It should small in size.
⮚ It should be easily introduced into the
host cell.
⮚ It should have the ability to replicate
autonomously in the host cell.
⮚ It must also be compatible with the
host organism.
⮚ The vector should have marker site
that allow easy detection of transformed cell.
⮚ It must possess a restriction site.
TYPES OF CLONING VECTOR
o Plasmids
o Bacteriophages
o Cosmids
o Artificial chromosomes
o Plasmids
Extra-chromosomal genetic material found in the cytoplasm of bacterial cell is known as
plasmid.
P Plasmid carries genes for antibiotic resistance but do not have any important functions.
A
G
E
Notes by S.D Sir
, *
M
E STUDY OF CLONING VECTORS, RESTRICTION ENDONUCLEASES AND DNA LIGASE
R
G
E ✔ Discovered by Hays & Lederberg.
F
O ✔ Plasmid are circular, double stranded and spiral molecules.
R
M ✔ Plasmid are capable of Independent
A Replication.
T
✔ Plasmid are not essential for the growth and
2 survival of bacteria.
✔ Plasmid has restriction site and marker gene
(antibiotic resistance gene).
Feature that are required to facilitate cloning:
1. ORI → Origin of replication. This
makes autonomous replication in vector.
2. Cloning Site (Restriction site) →
Cloning site is a place where we can
make a cut by restriction site and foreign
DNA can be added.
3. Selectable marker → A gene that
helps to select the transformed cell with
desired gene from non-transformed cell.
So, gene is marker gene.
▪ Nomenclature of plasmid cloning vector: pBR322 cloning vector has the following
elements:
● p= Plasmid
● B= Bolivar (name of the scientist)
● R= Rodriguez (name of the scientist)
● 322= Number given to distinguish
this from other developed from
same laboratory.
“First genetically engineered plasmid
widely used in rDNA technology”
It has;
P i. ORI (Origin of replication).
A
G
E
Notes by S.D Sir
