Download Cloning Vector

Presentation Topic
Cloning Vector and its Types
Presented By
Farah Mushtaq 117117
Faiza Saddique 117119
CLONING VECTORS
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A DNA molecule that carries foreign DNA
into a host cell, replicates inside a bacterial
(or yeast) cell and produces many copies of
itself and the foreign DNA
PLASMID
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Bacterial cells may contain
extra-chromosomal DNA
called plasmids.
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Plasmids are usually
represented by small,
circular DNA.
Some plasmids are
present in multiple copies
in the cell
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PLASMID VECTORS
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Plasmid vectors are ≈1.2–
3kb and contain:
replication origin (ORI)
sequence
a gene that permits
selection,
Here the selective gene is
ampr; it encodes the enzyme
b-lactamase, which
inactivates ampicillin.
Exogenous DNA can be
inserted into the bracketed
region .
TWO MAIN FEATURES OF THE CLONING
VECTOR
ORIGIN OF REPLICATION
 SELECTABLE MARKER
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ORIGIN OF REPLICATION
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Origin of replication is
a DNA segment
recognized by the
cellular DNA-replication
enzymes. Without
replication origin, DNA
cannot be replicated in
the cell.
SELECTIVE MARKER
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Selective marker is required
for maintenance of plasmid in
the cell.
Because of the presence of the
selective marker the plasmid
becomes useful for the cell.
Under the selective conditions,
only cells that contain plasmids
with selectable marker can
survive
Genes that confer resistance
to various antibiotics are used.
Genes that make cells
resistant to ampicillin,
neomycin, are used
MULTIPLE CLONING SITE
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Many cloning vectors contain a
multiple cloning site or
polylinker: a DNA segment
with several unique sites for
restriction endo- nucleases
located next to each other
Restriction sites of the
polylinker are not present
anywhere else in the plasmid.
Cutting plasmids with one of
the restriction enzymes that
recognize a site in the
polylinker does not disrupt any
of the essential features of the
vector
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Gene to be cloned can
be introduced into the
cloning vector at one of
the restriction sites
present in the
polylinker
TYPES OF CLONING VECTORS
CLONING VECTORS
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The vector is chosen according to the size
and type of DNA to be cloned Different
types of cloning vectors are used for
different types of cloning experiments.
PLASMID VECTORS
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Plasmid vectors are
used to clone DNA
ranging in size from
several base pairs to
several thousands of
base pairs (100bp 10kb).
ADVANTAGES OF PLASMIDS
small size (easy to manipulate and isolate)
 circular (more stable)
 replication independent of host cell
 several copies may be present (facilitates
replication)
 frequently have antibody resistance
(detection easy)
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DISADVANTAGES USING PLASMIDS
 range
from 0- 10 kb
 Standard methods of transformation
Cannot accept large fragments
 Sizes are inefficient
BACTERIOPHAGE LAMBDA
It infects bacteria
 Lambda phage vector
 High transformation efficiency, about 1000
times more than the plasmids vector
 Origin of replication
 Genes for head and tail protein
 Single-stranded producing cohesive ends
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BACTERIOPHAGE LAMBDA
YEAST ARTIFICIAL CHROMOSOMES
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Purpose:
Cloning vehicles that propogate in eukaryotic cell
hosts as eukaryotic Chromosomes
Clone very large inserts of DNA: 100 kb - 10 Mb
Features:
YAC cloning vehicles are plasmids
Final chimeric DNA is a linear DNA molecule with
telomeric ends: Artificial Chromosome
Additional features:
 Often have a selection for an insert
 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
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RETROVIRAL VECTORS
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Retroviral vectors are used to introduce new or altered
genes into the genomes of human and animal cells.
Retroviruses are RNA viruses.
The viral RNA is converted into DNA by the viral reverse
transcriptase and then is efficiently integrated into the host
genome
Any foreign or mutated host gene introduced into the
retroviral genome will be integrated into the host
chromosome and can reside there practically indefinitely.
Retroviral vectors are widely used to study oncogenes and
other human genes.
EXPRESSION VECTORS
Allows a cloned segment of DNA to be
translated into protein inside a bacterial
or eukaryotic cell.
 Vectors will contain the ff:
 (a) in vivo promoter
 (b) Ampicillin selection
 (c) Sequencing primers
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 Produces
large amounts of a specific
protein
 Permits studies of the structure and
function of proteins
 Can be useful when proteins are rare
cellular components or difficult to isolate
REPORTER GENE VECTORS
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A gene that encodes a protein whose activity
can be easily assayed in a cell in which it is not
normally expressed
These genes are linked to regulatory sequences
whose function is being tested
Changes in transcriptional activity from the
regulatory sequences are detected by changes
in the level of reporter gene expression
SHUTTLE VECTORS
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Shuttle vectors can replicate in two different
organisms, e.g. bacteria and yeast, or mammalian
cells and bacteria.
They have the appropriate origins of replication.
Hence one can clone a gene in bacteria, maybe
modify it or mutate it in bacteria, and test its function
by introducing it into yeast or animal cells
COSMID VECTOR
Combine parts of lambda chromosomes with
parts of plasmids
 An origin of replication(ori)
 A cos site (a sequence yield cohesive ends)
 An amplicine resistance gene (amp)
 Restriction sites for cloning
 Cosmids carry up to 50 kb of inserted DNA
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APPLICATIONS
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A particular gene can be isolated and its nucleotide
sequence determined
Control sequence of DNA can be identified and
analyzed
Protein/enzyme/RNA functions can be investigated
Mutation can be identified, e.g. gene defects related
to specific diseases
Organisms can be engineered for specific purposes,
e.g. insulin production
SUMMARY
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Cloning vector - a DNA molecule that carries foreign DNA into a host cell,
replicates inside a bacterial (or yeast) cell and produces many copies of
itself and the foreign DNA
Three features of all cloning vectors
sequences that permit the propagation of itself in bacteria (or in yeast for
YACs)
a cloning site to insert foreign DNA; the most versatile vectors contain a site
that can be cut by many restriction enzymes
a method of selecting for bacteria (or yeast for YACs) containing a vector
with foreign DNA; uually accomplished by selectable markers for drug
resistance
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General Steps of Cloning with Any Vector
prepare the vector and DNA to be cloned by digestion
with restriction enzymes to generate complementary
ends
ligate the foreign DNA into the vector with the enzyme
DNA ligase
introduce the DNA into bacterial cells (or yeast cells
for YACs) by transformation
select cells containing foreign DNA by screening for
selectable markers (usually drug resistance)
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