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Cloning Vectors-WPS Office

A cloning vector is a small piece of DNA that can stably maintain foreign DNA fragments inserted into it. Common cloning vectors include plasmids from bacteria, viruses like phage λ, and artificial chromosomes. The vector and insert DNA are cut with restriction enzymes, generating compatible sticky or blunt ends, and then joined together with ligase. Vectors contain features like selectable markers and origins of replication to be propagated in host cells like E. coli. Different vectors are suitable for different sized inserts and applications.

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56 views

Cloning Vectors-WPS Office

A cloning vector is a small piece of DNA that can stably maintain foreign DNA fragments inserted into it. Common cloning vectors include plasmids from bacteria, viruses like phage λ, and artificial chromosomes. The vector and insert DNA are cut with restriction enzymes, generating compatible sticky or blunt ends, and then joined together with ligase. Vectors contain features like selectable markers and origins of replication to be propagated in host cells like E. coli. Different vectors are suitable for different sized inserts and applications.

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deepak gupta
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Cloning Vectors

By Dr Deepak Rajpurohit
A cloning vector is a small piece of DNA that can be
stably maintained in an organism, and into which a
foreign DNA fragment can be inserted for cloning
purposes.The cloning vector may be DNA taken from a
virus, the cell of a higher organism, or it may be the
plasmid of a bacterium. The vector therefore contains
features that allow for the convenient insertion or
removal of a DNA fragment to or from the vector, for
example by treating the vector and the foreign DNA with
a restriction enzyme that cuts the DNA. DNA fragments
thus generated contain either blunt ends or overhangs
known as sticky ends, and vector DNA and foreign DNA
with compatible ends can then be joined together by
molecular ligation
DNA fragments thus generated contain either blunt ends or
overhangs known as sticky ends, and vector DNA and foreign
DNA with compatible ends can then be joined together by
molecular ligation. After a DNA fragment has been cloned
nto a cloning vector, it may be further subcloned into
another vector designed for more specific use.
There are many types of cloning vectors, but the most
commonly used ones are genetically engineered plasmids.
Cloning is generally first performed using Escherichia coli, and
cloning vectors in E. coli include plasmids, bacteriophages
(such as phage λ), cosmids, and bacterial artificial
chromosomes (BACs). Some DNA, however, cannot be stably
maintained in E. coli, for example very large DNA fragments,
and other organisms such as yeast may be used.
Features
All commonly used cloning vectors in molecular
biology have key features necessary for their function,
such as a suitable cloning site and selectable marker.
Others may have additional features specific to their
use. For reason of ease and convenience, cloning is
often performed using E. coli. Thus, the cloning vectors
used often have elements necessary for their
propagation and maintenance in E. coli, such as a
functional origin of replication (ori). The ColE1 origin of
replication is found in many plasmids. Some vectors
also include elements that allow them to be
maintained in another organism in addition to E. coli,
and these vectors are called shuttle vector.
Types
A large number of cloning vectors are available,
and choosing the vector may depend a number of
factors, such as the size of the insert, copy number
and cloning method. Large insert may not be
stably maintained in a general cloning vector,
especially for those with a high copy number,
therefore cloning large fragments may require
more specialized cloning vector.
Plasmids
Plasmids are autonomously replicating circular extra-
chromosomal DNA. They are the standard cloning vectors
and the ones most commonly used. Most general
plasmids may be used to clone DNA insert of up to 15 kb
in size. One of the earliest commonly used cloning vectors
is the pBR322 plasmid. Other cloning vectors include the
pUC series of plasmids, and a large number of different
cloning plasmid vectors are available. Many plasmids have
high copy number, for example pUC19 which has a copy
number of 500-700 copies per cell,[14] and high copy
number is useful as it produces greater yield of
recombinant plasmid for subsequent manipulation.
However low-copy-number plasmids may be preferably
used in certain circumstances, for example, when the
protein from the cloned gene is toxic to the cells.
Bacteriophage
The bacteriophages used for cloning are the phage λ and
M13 phage. There is an upper limit on the amount of DNA
that can be packed into a phage (a maximum of 53 kb),
therefore to allows foreign DNA to be inserted into phage
DNA, phage cloning vectors may need to have some non-
essential genes deleted, for example the genes for lysogeny
since using phage λ as a cloning vector involves only the
lytic cycle.[16] There are two kinds of λ phage vectors -
insertion vector and replacement vector. Insertion vectors
contain a unique cleavage site whereby foreign DNA with
size of 5–11 kb may be inserted. In replacement vectors, the
cleavage sites flank a region containing genes not essential
for the lytic cycle, and this region may be deleted and
replaced by the DNA insert in the cloning process, and a
larger sized DNA of 8–24 kb may be inserted.
Cosmids
Cosmids are plasmids that incorporate a
segment of bacteriophage λ DNA that has the
cohesive end site (cos) which contains
elements required for packaging DNA into λ
particles. It is normally used to clone large
DNA fragments between 28 and 45 Kb.[13]
Human Artificial ch
romosome
Human artificial chromosome may be potentially
useful as a gene transfer vectors for gene delivery
into human cells, and a tool for expression studies
and determining human chromosome function. It
can carry very large DNA fragment (there is no
upper limit on size for practical purposes),
therefore it does not have the problem of limited
cloning capacity of other vectors, and it also avoids
possible insertional mutagenesis caused by
integration into host chromosomes by viral vector.
Questions???

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