2. 2
The cloning of a gene by the method described by
Stanley Cohen and coworkers (1973) requires the use
of a suitable gene cloning vector.
A gene cloning vector should have capability of
autonomous replication, small size, origin of
replication, presence of a selectable marker gene(s)
and presence of unique restriction enzyme site(s).
In the first gene cloning experiment plasmid pSC101
was used for cloning of E. coli kanamycin resistant
gene. Natural plasmids are not suitable vectors for
gene cloning.
3. 3
•Several plasmid vectors like Col E1, pBR322, pUC series and
pGEMR series have been developed to suit the various requirements.
•It is not easy to clone a DNA fragment larger than 10 kb using a
plasmid vector as the transformation frequency decreases
considerably with the increased size of the recombinant plasmid. To
overcome this difficulty three research groups independently reported
the construction of bacteriophage lambda cloning vectors in 1974.
•Subsequently, to meet the various requirements of cloning, the
following vectors were constructed in the respective years mentioned
in the brackets: Bacteriophage M13 derivatives (1977), cosmids
(1978), yeast artificial chromosomes (1987), bacteriophage P1
(1990), bacterial artificial chromosomes (1992), P1 artificial
chromosomes (1994), human artificial chromosomes (1997) and
maize mini chromosomes (2007).
4. Properties and construction of a vector DNA molecule
1. Capability of autonomous replication
• Bacterial and viral genomes contain only one origin of
replication while eukaryotes contain multiple origins
2. Small size
• In small molecules the chances of occurrence of unique sites
for restriction enzymes increases
• Efficiency of gene transfer is high with small vector
molecules
3. Presence of selectable marker gene(s)
• For easy detection of recombinants
• E.g.: antibiotic resistant genes, lac z or resistant to toxins, etc
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5. 4. Presence of unique restriction enzyme sites or
multiple cloning sites for inserting the target DNA
• Position of these restriction sites should be such that the
insertion of a segment of DNA in any of these restriction sites
bring about a phenotypic change in the characteristic of a vector
molecule e.g.: loss of gene expression or loss of resistance to an
antibiotic
5.Ease of purification
6. No effect on the replicative ability of vector due to
insertion of target DNA
7. Ease of reintroduction into host cell with high
efficiency
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6. 8. Biological containment
•Vectors should be biologically contained with no possibility
of gene escape
•This can be achieved by non-conjugative and non-mobilized
plasmid vectors
9.Presence of promoters and ribosome binding
sites
10. Presence of two different origins of replication
or broad host range origin of replication
E.g.: shuttle vectors that contain two different origins of
replication
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8. 1. Generation of the DNA fragment
2. Construction of the recombinant DNA
molecule by joining of DNA fragment with a
vector
3. Introduction of the recombinant vector into
host cell, multiplication of recombinant DNA
molecule along with host cell
Steps involved in gene cloning
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9. Plasmid characteristics
1. Capability of autonomous replication
2. Small size
3. Presence of selectable marker gene(s)
4. Presence of unique restriction enzyme sites
5. Non-conjugative and non-mobilizable
6. Replicon under relaxed control
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10. Natural plasmid vectors for E. coli
pSC101
• First used for in vitro cloning of eukaryotic DNA
• 9kbp in size
• Low copy number(1-2 copies)
• Has advantage of a single Eco RI site at which DNA can be
inserted
• Has selectable marker for tetracycline resistance
• Derived from the conjugative plasmid R6-5
Disadvantages are
• Large size
• Stringent replicative control
• Low copy number
• Low insert capability
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12. pSF 2124( RSF2124)
• Produced by transfer of the ampicillin resistance gene
•Has ability for colicin biosynthesis
•Has high copy number
•Has single sites for Bam H1 and EcoR1
• Not currently used as vector as it does not provide easy
selection by insertional inactivation
•Mobilizable plasmid
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13. Col E1 Plasmid
• Small, circular colicingenic plasmid
•Codes for 57kDa protein toxin
•Size is 6,466bp
•Has cea gene for colicin production
• imm for immunity against colicin
•kill for killer or lysis protein
•mob for mobilization
13
16. •pBR322 is a plasmid and was the first widely-used E.
coli cloning vector
•Created in 1977, it was named after its Mexican
creators, p standing for plasmid, and BR for Bolivar
and Rodriguez
•pBR322 is 4361 base pairs in length and contains a
replicon region (source plasmid pMB1)
•The ampR gene, encoding the ampicillin
resistance protein (source plasmid RSF2124)
•The tetR gene, encoding the tetracycline resistance
protein (source plasmid pSC101 )
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17. •Has unique restriction sites for more than 40
restriction enzymes
•11 of these 40 sites lie within the tetR gene
•Has 2 sites for restriction enzymes
HindIII and ClaI within the promoter of the tetR
gene
•Six key restriction sites inside the ampR gene
•The origin of replication or ori site in this plasmid
is pMB1 (a close relative of ColE1)
•The ori encodes two RNAs (RNAI and RNAII) and
one protein (called Rom or Rop )
17
19. pUC series
•pUC19 is one of a series of
plasmid cloning vectors
created by Messing and co-
workers in the University of
California..
•The p in its name stands for
plasmid and UC represents
the University in which it was
created. It is a circular double
stranded DNA and has 2686
base pairs
•Series include pUC 18 pUC
19, pUC12 and pUC 13, etc
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20. •It has one ampR gene (ampicillin resistance gene)
•Has an N-terminal fragment of β-galactosidase (lac Z ) gene of E.
coli
•The multiple cloning site (MCS) region is split into the lac Z gene
(codons 6–7 of lac Z are replaced by MCS), where
various restriction sites for many restriction endonucleases are
present
•The ori site or replicon, rep is derived from pMB1 vector
•pUC vector is small but has a high copy number. The high copy
number of pUC plasmids is a result of the lack of the rop gene
and a single point mutation in rep of pMB1
•The lac Z gene codes for β-galactosidase
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22. •The pGEM-3Z Vector is intended for use as a standard cloning
vector, as well as for the highly efficient synthesis of RNA in
vitro
•The vector carries the lacZ α-peptide and the multiple cloning
region arrangement from pUC18 allowing selection of
recombinants by blue/white screening
•In addition, the vector contains both the SP6 and T7 RNA
polymerase promoters flanking the multiple cloning region
•The pGEM-3Z and pGEM-4Z Vectors are essentially identical
except for the orientation of the SP6 and T7 promoters
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24. • The pET-28a-c(+) vectors are most powerful
for cloning and expression of recombinant
proteins in E. coli.
• The pET-28a-c(+) vectors carry an N-terminal
His•Tag®/thrombin/T7•Tag® configuration
plus an optional C-terminal His•Tag sequence.
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