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What is cloning?
The isolation of discrete pieces of DNA from their host organism and their
amplification through propagation in the same or a different host
More recently an alternitive, in vitro method for amplifying a paticular
DNA segment has been developed: polymerase chain reaction PCR
Why cloning?
The molecular cloning process makes it possible to study in detail the
structure and functioning of genes and other DNA sequences from even
the most complex genomes
Provides a method for purifying a large quantity of a specific DNA for
analysis
Components of cloning
DNA to be cloned
Genomic DNA
cDNA
Any artificail DNA
Cloning vector
Plasmids
Viral DNA-s bacteriophage , filamentous bacteriophages
Hybrid vectors cosmids, phageimids
Cells
Select a source of DNA for cloning which contains the sequence of interest
Prepare DNA of appropriate length and suitably structured on each end joining with
the cloning vector
Select a cloning vector and prepare it to receive the DNA to be cloned so that the
insertion of foreign DNA to the vector does not diminish its capacity as an
independent replicon:
use restriction enzyme(s)
Modify ends of prepared DNA to be cloned to make it compatible for joining with the
vector:
use restriction enzyme(s)
Join in vitro the molecules of vector DNA and DNA to be cloned to form recombinant
DNA molecules:
use DNA ligase
Introduce the recombinant DNA molecules, one per cell, into viable host cells
capable of replicating the vector from which the recombinant DNA was prepared:
transform E.Coli cells
Screen the collection of recombinant vectors to identify those carrying the cloned
DNA sequence and propagate them as clones
Confirm the identity of the selected clones by different ways: restriction analysis
and/or PCR.
Most naturally occuring plasmids confer to a host cell a distinct phenotype, such as
fertility, drug resistance, heavy metal tolerance, etc.:
The genetic determinants encoded by plasmids enable their bacterial
host to survive better in adverse enviroments
Plasmids are rarely essential for bacterial survival
Antibiotic-resistance gene
(ampicillin)
Range in size from 1 to more than 200 kb, most often exist as double-stranded
circular DNA molecules, but their conformation can vary:
Relaxed, covalently closed circular DNA
Supercoiled DNA
Open, circular DNA
PLASMID CONFORMATIONS
DNA gyrase
Endonucleas
e
Topoisomerase
Endonucleas
e
DNA ligase
Cloning a foreign DNA into SalI site results in insertional inactivation of an tetracycline
resistance gene:
-
transformants are first selected by plating the transformed E.Coli cells to the
ampicillin-containing plate
only the cells containing plasmid with or without the insert will
grow
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Makes use of the lac operon of E.Coli that encodes proteins necessary for the
utilization of lactose:
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Plasmid lacZ region is mutated like this that vector has only a partial lac operon of
E.Coli containing the promoter which encodes 146 amino acid long -peptide (the Nterminal part of -Galatosidase).
Host E.Coli strain has also mutation in its genome which encodes only the other half
(lacking N-terminus) of -Galatosidase -complemententing fragment.
-peptide and -fragment are not active by themselves but when combined they
generate a functional enzyme molecule.
A multiple cloning site is inserted into the 5 end of the -fragment in the vector so
that the reading frame of the -fragment is intact
Cloning of a DNA into the -fragment using some of the enzymes present in the
multiple cloning site will inactivate the gene, allowing to screen for insertion of a
clone by looking for a lactose-negative phenotype:
Plasmids found in the white colonies contain an insert and can be
easily identified from the background blue colonies that have plasmids
without insert.
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Insert DNA
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pUC FAMILY
Genetic maps of some pUC plasmids. The multiple cloning site (MCS) is inserted into
the lacZ gene but does not interfere with gene function.
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Some plasmids are designed to express the gene product encoded in the foreign
DNA, usually as fusion protein with an E.Coli gene product:
Example in pUC18 the lacZ gene construct functions to produce -
Some plasmid vector of later generation have additional features which simplify later
manipulations:
Example: different bacteriophhage promoter sequences (SP6, T3, T7)
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In the case of some genes they can not be used since the metabolic load inflicted on
the host cell by massive production of a recombinant DNA or expression of a foreign
protein may result in undesirable instabilities, plasmid loss or even cell death.
Low copy number plasmids
Avoids deleterious effects of high copy number plasmids, but may create other
problems since the amount of the recombinant DNA will be low and the expression
of the cloned gene will be reduced.
Runaway plasmid vectors
At 30C the plasmid vector is present in a moderate number of copies per cell
Above 35C the control of plasmid replication is lost and the number of plasmid
copies per cell increases continuosly
At the higher temperature cells grow normally for 2-3 hours, and the products from
genes on the plasmid are over-produced
Finally, cells die, but at this stage plasmid DNA may account for as much as 50% of
total DNA in the cell.
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PROS
-
The small size of the vector DNA simplifies the purification and analysis of the foreign
DNA
Large quantities of foreign DNA may be produced easily using a plasmid vector
CONS
-
Joining foreign DNA to vector leads to recombinant molecules with disappointing low
transformation efficiencies
Plasmids in general are poor vectors of large foreign DNA fragments (>10 kb), which
is due to lower efficiency of circular plasmid formation during ligation and to a lower
probability of transforming competent cells
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BACTERIOPHAGE
(DOUBLE-STRANDED DNA VECTOR)
particles consist of a head, which contains DNA, and a tail, which is used for cell
adsorption:
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GENOME
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After has infected a bacterium, two developmental pathways are open to the virus:
The lytic pathway were the viral functions are fully expressed, leading to the lysis of
the bacterium and the appearance of about 100 progeny virus particles.
The goal of producing a large number of progeny is achived by the
sequential transcription of viral genes: early stage establishes the
lytic cycle; middle stage proteins neseccary for the replication of
DNA and for recombinant are made; late stage proteins that
compose the head and tail of the phage and those that lyse the host
are made.
The lysogenic pathway were the genome becomes covalently inserted into the host
cell chromosome through recomnbination at a specific site.
Most of the phage functions are turned off and the viral DNA in this
stage is called a prophage, a part of the chromosome that is replicated
along with the bacterial DNA
Under the appropriate environmental stimuli, usually some form of
damage to the host chromosome such as exposure to UV light, the
prophage may excise from the chromosome and replicate via the lytic
pathway.
In this state only gene expressed is cl (look next slide) encoding the
repressor which binds to two operator regions OL and OR.
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LYTIC PATHWAY
LYSOGENIC PATHWAY
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Only about 50% of the genes in the genome are requiered for lytic growth
The nonessential genes, clustered in the middle of the genome are
The packaging mechanism that inserts DNA into the phage head has a stringent requierment
for DNA size
DNA may be packed into infectious phage particles in vitro using mixed extracts of
cells carrying defective strain as prophages
recombinant DNA packaged into phage particles may be introduced into E.Coli 10100 times more efficiently thanrecombinant plasmid DNA.
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Insertion vectors - a foreign DNA is cloned into the vector at a single unique restriction site
Frequently used for cDNA cloning and applications that requier small
DNA fragments.
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PROS
-
unamplified libraries are easily stored for long periods at 4 C due to the stability of
the phage particles.
CONS
-
The large size (24 kb) of the arms complicates the restriction analysis of foreign
DNA.
It is more difficult to produce large quantities of phage DNA than plasmid DNA
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COSMID VECTORS
(DOUBLE-STRANDED DNA VECTOR; HYBRID)
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____________________________________________________________________
In
vitro
concatemerized
packaging
phage-
Mature phage
particles
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FILAMENTOS PHAGE
(SINGLE-STRANDED DNA VECTOR)
M13, f1 and fd
Contain single-stranded DNA of about 6.4 kb, which is protected by a coat of 2710
identical protein subunits
Filamentous phages can only infect bacteria harbouring F pili encoded by F episome.
F pilus is encoded by the F episome that is integrated into the E.Coli
chromosome
The F episome can also be maintained as as plasmid called F
E.Coli that contain F as a plasmid or integrated into the chromosome
are capable of making the F pilus
F plasmid can be lost at a fairly high frequency it is important to
maintain a selection for the F plasmid. This can be accomplished by
having a selectable marker on the plasmid
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- Unlike phage , filamentous phages do not have any non-essential genes, which can
be used as cloning sites
- M13 has 507 bp inergenic region containing the origins of replication which can be
used to insert foreign DNA
- The wild type M13 is not very useful as a vector since it contains very few unique
restriction sites in the intergenic region
- Several M13-based vector have been constructed containing polycloning sites with
suitable restriction sites
- E.Coli lac regulatory region encoding -galactosidase -peptide has been introduced
into the intergenic region and polycloning site has been inserted into its coding
sequence, allowing to select for recombinant plaques by blue/white screen on X-gal
plates.
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PHAGEMIDS
(SINGLE-STRANDED DNA VECTOR; HYBRID)
- A hybrid vector containing plasmid replication genes and replication elements from
the filamentous phage
The intergenic region of the f1 filamentous phage contains all of th
signals requiered for packaging and DNA replication
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- The introduction of the intergenic region of the filamentous phage into plasmid
vectors allows them to replicate either as a plasmid or as a phage
The phage origin is inactive during propagation of double-stranded
phagemid by the plasmid origin since gene 2 of the filamentous phage
is not present: the vector behaves as a plasmid with double-strand
replication allowing to clone and stably maintain large DNA fragments
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Plasmid vectors
- Relatively small size makes them easy to use, purify, and analyze
- Depending on the foreign DNA to be cloned, plasmids with different copy numbers
can be used: high and low copy number, runaway type
- High efficiency of preparing recombinant clones due to the efficient packaging and
delivery mechanism
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