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Cloning Vector: BY: Dr. Mukesh Kumar Bhardwaj

Cloning vectors are DNA molecules that can accept foreign DNA and replicate within a host cell. They must be able to replicate autonomously and have selectable markers and restriction sites. Common types include plasmid vectors, bacteriophage vectors, cosmid vectors, phagemid vectors, and artificial chromosome vectors. Plasmid vectors like pBR322 are small, circular, and can replicate in bacteria. Bacteriophage vectors like lambda can package large DNA fragments and have high transformation efficiency. Applications include gene isolation, sequence analysis, and engineering organisms to produce proteins.

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محمود محمد
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96 views

Cloning Vector: BY: Dr. Mukesh Kumar Bhardwaj

Cloning vectors are DNA molecules that can accept foreign DNA and replicate within a host cell. They must be able to replicate autonomously and have selectable markers and restriction sites. Common types include plasmid vectors, bacteriophage vectors, cosmid vectors, phagemid vectors, and artificial chromosome vectors. Plasmid vectors like pBR322 are small, circular, and can replicate in bacteria. Bacteriophage vectors like lambda can package large DNA fragments and have high transformation efficiency. Applications include gene isolation, sequence analysis, and engineering organisms to produce proteins.

Uploaded by

محمود محمد
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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CLONING VECTOR

 BY:
 DR. MUKESH KUMAR BHARDWAJ
WHAT IS CLONING VECTOR ?

 A cloning vector is a DNA molecule in which foreign DNA can be inserted or


integrated and which is further capable of replicating within host cell to
produce multiple clones of recombinant DNA.
CHARACTERISTICS

 It should be able to replicate autonomously.


 Origin of replication
 Selectable markers
 Restriction sites
TYPES

 Plasmid Vector
 Bacteriophage Vector
 Cosmid Vector
 Phagemid Vector
 Phasmid Vector
 Artificial Chromosome Vector
1. PLASMID VECTOR

 Plasmid is a DNA molecule , other than


the artificial chromosome .
 Plasmid is a circular and double
stranded DNA molecule and size
ranges from 1 kb to over 250 kb.
 The plasmids can be single copy
plasmids that are maintained as 1
plasmid DNA per cell or multicopy
plasmids which are maintained 10-20
genome per cell.
TYPES OF PLASMID VECTOR

1. pBR322
2. pBR327
3. pUC
4. pSP64,pSP65
5. Agrobacterium plasmid vector
1.pBR322

Contains:-
q Selectable markers –
§ Ampicillin resistance gene
§ Tetracyclin resistance gene
§ Col E1 replication origin
§ Eco R1 site
2. pBR327

 It is derived from pBR322 by deletion of


nucleotides between 1427 to 2516.
 These nucleotides are deleted to reduce
the size of vector and eliminate sequences
that were known to interfere with the
expression of cloned DNA in eukaryotic
cell.
 It contains genes for resistance against
two antibiotics .
3. pUC Vector

Contains :-
q Ampicillin resistance gene
q Multiple cloning site
q Col E1 (Origin)
4. pSP64,pSP65

 These vectors are derivatives of pUC


plasmid vector and carry promoters for
phage RNA polymerase to allow
transcription in vitro .
 pSP64 and pSP65 to contain the phage
promoter SP6 and the poly cloning
sites arranged in opposite orientation to
allow transcription of desired strand
from the cloned double stranded DNA.
5. Agrobacterium Plasmid Vector

 In higher plants Ti plasmid of


Agrobacterium tumifacience or
Ri plasmid of Agrobacterium
rhizogene are best known
vectors.
 T- DNA from Ti or Ri plasmid
of Agrobacterium is considered
to be a very potential vector for
cloning experiment.
BACTERIOPHAGE VECTOR

 Bacteriophages are
viruses that attack
bacteria .
 Several bacteriophages
like Lambda (ʎ) and
M13 are used as
cloning vectors .
Lambda (ʎ) phage Vector:-

 High transformation efficiency ,


about 1000 times more efficient
than plasmid vector.
 Origin of replication
 Genes for head and tail proteins
 Single stranded protruding
cohesive ends
 Size is 48 - 502 bp
Ø
Phage M13 Vector

 These vectors are used for obtaining single


stranded copies of cloned DNA which are
specially suited for DNA sequencing .
 They are derived from 6.4kb genome of
E.coli , filamentous bacteriophage M13.
 This phage has a single stranded , linear
DNA genome in phage particle which
becomes converted into a double stranded ,
circular replicative intermediate with in the
host cell
3. COSMID VECTOR

 Combine part of the Lambda


chromosome with parts of plasmids.
 An origin of replication (Ori).
 A cos site (a sequence yield cohesive
end )
 An ampicillin resistance gene (amp)
 Restriction site for cloning .
 Cosmid can carry up to 50 kb of
inserted DNA.
4. Phagemid Vectors

 A plasmid vector that contains origin of


replication from a phage, in addition to
that of plasmid is called Phagemid.
 P Blue script SK is a phagemid vector of
2958 base pairs derived from pUC19 . It
contain ;
1. Phage N13 origin of replication
2. A portion of lac Z gene driven by lac
promotor.
3. A multi[ple cloning site (MCS) within
lac Z gene .
5. Phasmid Vector

 These vectors are ʎ insertion vectors


consisting of shortened linear ʎ genome
containing DNA replication and lytic function
+ the cohesive end of phase .
 A good example of phasmid ʎ ZAP. The main
features of ʎ ZAP are as below:-
i. The DNA insert is placed with MCS located
in the lac Z gene of P blue script SK(-).
ii. It is suitable for cloning of cDNA are using
EcoR1 linker.
iii. Integration of DNA insert inactivates lac Z
6. Artificial chromosome vector

 These vectors are circular or linear vector that are stably maintain usually 1-
2 copies per cell.
 There are several types of such vectors ;
1. Yeast Artificial Chromosome [YAC]
2. Bacterial Artificial Chromosome [BAC]
3. Mammalian Artificial Chromosome [MAC]
4.Humun Artificial Chromosome [HAC]
Yeast Artificial Chromosome

 YAC are genetically engineered


chromosome derived from the
DNA of the Yeast ,
Saccharomyces cerevisiae.
 YAC vectors allow the cloning,
within yeast cells , of fragments
of foreign genomic DNA that
can approach 500 kbp in size.
Bacterial Artificial Chromosome

 A BAC is a DNA construct,


based on a functional fertility
plasmid , used for
transforming and cloning in
bacteria , usually E.Coli.
 They are capable of carrying
approximate up to 300 kbp of
inserted DNA sequence.
Mammalian Artificial Chromosome

 MAC are produced


by mammalian
telomere and some
centromeric
sequences.
Human Artificial Chromosome

 A HAC is a microchromosome
that can act as a new chromosome
in a population of humun cells.
 That is , instead of 46
chromosome, the cells could have
47 with the 47 th being very
small , roughly 6-10 megabases
(Mb) in size instead of 50-250 Mb
for natural chromosomes , and
able to carry new genes
introduced by humun researchers.
Applications

 A particular gene can be isolated and its nucleotide sequence is determined.


 Control sequence of DNA can be identified and analysed.
 Protein / Enzyme / RNA function can be investigated .
 Mutation can be identified example gene defects related to specific disease.
 Organisms can be engineered for specific purposes example Insulin
production

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