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Cloning Vector FINAL PPT

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1K views

Cloning Vector FINAL PPT

Uploaded by

AYESHA
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PPTX, PDF, TXT or read online on Scribd
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CLONING VECTOR

GROUP
MEMBERS:
ZAIN ALI AQSA ATHAR
SADAF AFTAB AREEJ MUSTAFA
ANZA NASEER SANA MUSTAFA
YUSRA MAJEED
Cloning vector:
The molecular analysis of DNA has been
made possible by the cloning of DNA. The
two molecules that are required for cloning
are the DNA to be cloned and a cloning
vector.
A cloning vector is a small piece of DNA
taken from a virus, a plasmid or the cell of
a higher organism, that can be stably
maintained in an organism and into which a
foreign DNA fragment can be inserted for
cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to
Cloning vector:
The vector therefore contains features that
allow for the convenient insertion or removal
of DNA fragment in or out of the vector, for
example by treating the vector and the
foreign DNA with a restriction enzyme and
then ligating the fragments together.
After a DNA fragment has been cloned into a
cloning vector, it may be further sub cloned
into another vector designed
for more specific use.
Cloning vector:
 Cloning vector is used as a vehicle to
artificially carry foreign genetic material
into another cell, where it can be replicated
and expressed.
It is used to amplify a single molecule of
DNA into many copes.
Cloning vectors are DNA molecules that are
used to "transport" cloned sequences
between biological hosts and the test tube.
Without Cloning Vector, Molecular Gene
Cloning is totally impossible.
FEATURES OF A CLONING
VECTOR:
 All commonly used cloning vectors have some essential
features:
Origin of replication (ori):
This makes autonomous replication in vector. – ori is a
specific sequence of nucleotide from where replication starts.
– When foreign DNA is linked to the sequence along with
vector replication, foreign (desirable) DNA also starts
replicating within host cell.
Cloning Site:
Cloning site is a place where the vector DNA can be
digested and desired DNA can be inserted by the same
restriction enzyme. – It is a point of entry or analysis for
genetic engineering work. – Recently recombinant plasmids
contain a multiple cloning site (MCS) which have many (up to
~20) restriction sites. Features of A Cloning Vector
FEATURES OF A CLONING
VECTOR:
Selectable Marker
Selectable marker is a gene that confers resistance
to particular antibiotics or selective agent that would
normally kill the host cell or prevent its growth. – A
cloning vector contains a selectable marker, which
confer on the host cell an ability to survive and
proliferate in a selective growth medium containing the
particular antibiotics.
Reporter Gene or Marker Gene
Reporter genes are used in cloning vectors to
facilitate the screening of successful clones by using
features of these genes that allow successful clone to be
easily identified. – Such feature present in cloning
vectors is used in blue- white selection.
Types of cloning vector:
 1. PLASMIDS:
 Plasmids were the first vectors to be used in gene cloning.
They are naturally occurring and autonomously replicating
extra- chromosomal double-stranded circular DNA
molecules. However, not all plasmids are circular in origin.
They are present in bacteria, archaea, and eukaryotes. The
size of plasmids ranges from 1.0 kb to 250 kb. DNA insert
of up to 10 kb can be cloned in the plasmids. The plasmids
have high copy number which is useful for production of
greater yield of recombinant plasmid for subsequent
experiments. The low copy number plasmids are exploited
under certain conditions like the cloned gene produces the
protein which is toxic to the cells. Plasmids only encode
those proteins which are essential for their own replication.
These protein-encoding genes are located near the ori.
pBR322 cloning vector:

 pBR322 is one of
the most
commonly
used E.coli cloning
vectors. pBR322 is
4361 bp in length.
Nomenclature

 The first letter is p means plasmid


 The second and third letters are capital
letters usually depict the name of the
institution or person who develop the
vector
 The fourth and the followers are roman
numerals were meant to indicate the
code used by the inventor. More often,
the roman numerals indicate the order
of discovery pBR322
Pros and Cons:
Advantages of using Disadvantages of using
Plasmids as vectors: Plasmids as vectors:
 Easy to manipulate and Large fragments
isolate because of small cannot be cloned.
size.
 More stable because of Size range is only 0
circular configuration. to 10kb.
 Replicate independent Standard methods
of the host. of transformation
 High copy number.
are inefficient.
 Detection easy because
of antibiotic-resistant
genes.
2. BACTERIOPHAGES
 Bacteriophages or phages are
viruses which infect bacterial
cells.
 The most common
bacteriophages utilized in
gene cloning are Phage λ and
M13 Phage.
 A maximum of 53 kb DNA can
be packaged into the phage.
 If the vector DNA is too small,
it cannot be packaged
properly into the phage.
 Examples: Phage Lambda,
M13 Phage, etc.
Phage lambda:
Phage lambda is a bacteriophage or phage, i.e. bacterial virus, that
uses E. coli as host. • Its structure is that of a typical phage: head,
tail, tail fibres. • Lambda viral genome: 48.5 kb DNA with a 12 base
ssDNA "sticky end" at both ends; these ends are complementary in
sequence and can hybridize to each other (this is the cos site:
cohesive ends).
Infection: lambda tail fibres adsorb to a cell surface receptor, the tail
contracts, and the DNA is injected. The DNA circularizes and lambda
begins its life cycle in the E. coli host.
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 may be deleted and replaced by
the DNA insert in the cloning process.
3= Phagemid:
A phagemid or phasmid is a
plasmid that contains an f1
origin of replication from an
f1 phage. It can be used as a
type of cloning vector in
combination with
filamentous phage M13. A
phagemid can be replicated
as a plasmid, and also be
packaged as single stranded
DNA in viral particles.
4= 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 25 and 45 Kb.
 They can replicate as plasmids if they
have a suitable origin of replication.
 They can also be packaged in phage
capsids, which allows the foreign genes
to be transferred into cells by
transduction.
 Advantages :
High transformation efficiency. The
cosmid vector can carry up to 45 kb
whereas plasmid and Lambda phage
5= yeast artificial chromosome
(YAC):
 The yeast artificial chromosome
(YAC) vector is capable of carrying
a large DNA fragment (up to 200
Kb), but its transformation
efficiency is very low.
 Cloning vehicles that propogate in
eukaryotic cell hosts as eukaryotic
chromosomes.
 Final chimeric DNA is a linear DNA
molecule with telomeric ends:
Artificial Chromosome.
 YAC cloning vehicles often have a
bacterial origin of DNA replication
(ori) and a selection marker for
propogation of the YAC through
bacteria.
 The YAC can use both yeast and
bacteria as a host.
6= Bacterial Artificial
Chromosome (BAC):
BAC vectors are similar to standard E. coli plasmid vectors.
Contain the origin and genes encoding the ori binding
proteins required for plasmid replication.
Derived from a naturally occurring large plasmid, the Fʹ
plasmid.
Low copy number (1-2 copies per cell)
The bacterial artificial chromosome's usual insert size is
150-350 kb.
BACs are preferred for different kind of genetic studies of
inherited or infectious diseases because they
accommodate much larger sequences without the risk of
rearrangement, and are therefore more stable than other
types of cloning vectors.
Cloning vectors
Vector system Host cell Insert capacity (kb)

Plasmid E. coli 0.1-10


Bacteriophage l E. coli 10-20

Cosmid E. coli 35-45

Bacteriophage P1 E. coli 80-100

BAC (bacterial E. coli 50-300


artificial
chromosome)
P1 bacteriophage- E. coli 100-300
derived AC
YAC Yeast 100-2,000
Human AC Cultured human >2,000

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