DNA CLONING

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DNA CLONING
DNA CLONING
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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
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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
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Components of cloning
 DNA to be cloned
Genomic DNA
cDNA
Any artificail DNA
 Cloning vector
Plasmids
Viral DNA-s – bacteriophage λ, filamentous bacteriophages
Hybrid vectors – cosmids, phageimids
 Cells
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ESSENTIAL STEPS IN MOLECULAR CLONING PROCESS
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Select a source of DNA for cloning which contains the sequence of interest
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Prepare DNA of appropriate length and suitably structured on each end joining with
the cloning vector
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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)
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Modify ends of prepared DNA to be cloned to make it compatible for joining with the
vector:
 use restriction enzyme(s)
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Join in vitro the molecules of vector DNA and DNA to be cloned to form recombinant
DNA molecules:
 use DNA ligase
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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
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Screen the collection of recombinant vectors to identify those carrying the cloned
DNA sequence and propagate them as clones
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Confirm the identity of the selected clones by different ways: restriction analysis
and/or PCR.
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GENERAL STEPS OF CLONING
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BASIC PROPERTIES OF PLASMIDS
(DOUBLE-STRANDED DNA VECTOR)
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Plasmids are extrachromosomal, self replicating, naturally occuring DNA molecules
that can be isolated as genetic entities.
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In E.Coli naturally occuring plasmids are doublestranded circular DNA molecules
which may be stably inherited by both daugther cells at cell division
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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)
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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
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PLASMID CONFORMATIONS
DNA gyrase
Endonucleas
e
Topoisomerase
Endonucleas
e
DNA ligase
Migration of plasmids on agarose gel
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PLASMID COPY NUMBERS
Depending to the type of replication, plasmids can be categorized into two classes:
 Non-conjugative - relaxed plasmids – maintained as multiple copies
per cell: usually 20-40, also called high-copy-number plasmids
 Conjugative - stringent plasmids – limited number of copies per cell:
usually 1-3, called low-copy-number plasmids
Conjugative plasmids enable bacteria to transfer genetic material to each other.
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PLASMIDS AS CLONING VEHICLES
Plasmids used as cloning vectors are derived from naturally occuring plasmids by
manipulating the genetic content of the plasmid in vitro to include at least 3 features:
 A replication origin for the propagation of the plasmid
 Gene(s) for selection
 Useful cloning sites, typically unique sites
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PROPERTIES OF AN „IDEAL“ ARTIFICIAL PLASMID CLONING VECTOR
Should express at least one selectable phenotype, usually drug resistance
 Since only about 10% of host cells accept and propagate a plasmid under
available transformation conditions, drug selection is used to kill host cells that
lack the plasmid after transformation; usually ampicillin and/or tetracylin
resistance genes are used in plasmid vectors
Should be small in size
 Easier to handle; small molecular weight plasmids are usually present as multiple
copies; higher chance for unique restriction sites
Should possess multiple cloning sites: usually unique restriction sites, where the circular
vector molecule may be opened to receive foreign DNA without loss of the capacity to
replicate or be selected:
 Usually a polylinker with 10 unique restriction sites is engineered to plasmid
vectors
Should possess one or more cloning sites arranged to display an identifiable phenotype
change as a result of insertion of foreign DNA:
 Insertional inactivation
 α-complementation
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INSERTIONAL INACTIVATION (1)
Cloning a foreign DNA into SalI site results in insertional inactivation of an tetracycline
resistance gene:
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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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replica plating onto tetracycline-containing media allows to select of transformants
containing an insert
 the cells that do not grow are recombinants
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INSERTIONAL INACTIVATION (2)
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α-COMPLEMENTATION OR BLUE-WHITE SCREENING (1)
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A direct selection of recombinants that provides a rapid visual detection of an insert
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Makes use of the lac operon of E.Coli that encodes proteins necessary for the
utilization of lactose:
or IPTG – isopropylthiogalactoside – a nonmetabolizable inducer (mimics lactose, but
not metabolized by bacteria)
β-Galatosidase is utilizing lactose by cleaving it into galactose and glucose.
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α-COMPLEMENTATION OR BLUE-WHITE SCREENING (2)
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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 α-peptide (the Nterminal part of β-Galatosidase).
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Host E.Coli strain has also mutation in its genome which encodes only the other half
(lacking N-terminus) of β-Galatosidase – ώ-complemententing fragment.
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α-peptide and ώ-fragment are not active by themselves but when combined they
generate a functional enzyme molecule.
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When E.Coli cell is transformed with a vector, enzymatically active β-galactosidase
accumulates when IPTG (mimics lactose) is included in the agar and transformed
colonies can be recognized by including in the agar a β-galactosidase chromogenic
substrate X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside):
 X-gal produces a blue precipitate when hydrolyzed by β-galactosidase
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A multiple cloning site is inserted into the 5´ end of the α-fragment in the vector so
that the reading frame of the α-fragment is intact
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Cloning of a DNA into the α-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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α-COMPLEMENTATION OR BLUE-WHITE SCREENING (3)
Insert DNA
Without insert DNA
With insert DNA --> recombinant
(minority)
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pUC FAMILY
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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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Different strains can bare different restriction enzymes.
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ADDITIONAL FEATURES OF PLASMID VECTORS (1)
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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 β-
Galatosidase fusion proteins when the foreign DNA is cloned in the
proper reading frame.
This feature is essential for preparing libraries when detection of the expressed gene
product forms the basis of the screening process
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Some plasmid vector of later generation have additional features which simplify later
manipulations:
 Example: different bacteriophhage promoter sequences (SP6, T3, T7)
have been introduced at positions flanking cloning sites which allows
to in vitro produce strand-specific RNA transcripts of the foreign DNA
from either strand for use in preparing hybridization probes, probes
for mobility shift electrophoresis, in vitro translation, sequencing or
the study of RNA processing.
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ADDITIONAL FEATURES OF PLASMID VECTORS (2)
Inclusion of the viral-specific promoter sequences from bacteriophage SP6 and/or T7 in a
cloning vector will allow one to obtain strand-specific transcripts from an inserted piece of
DNA. The SP6 RNA polymerase will specifically transcribe on strand of DNA from its
promoter. The oppositely oriented T7 promoter allows the T7 RNA polymerase to transcribe
the complementary strand.
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PLASMIDS WITH ALTERED COPY NUMBERS
High copy number plasmids
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Derived from plasmids exhibiting relaxed type of replication
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Copy number stands around 20 copies per cell
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Useful for synthesis of large amounts of plasmid DNA
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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
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Maintained as limited number copies per cell, usually 1-3
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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
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Makes use of temperature-sensitive origin of replication:
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At 30°C the plasmid vector is present in a moderate number of copies per cell
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Above 35°C the control of plasmid replication is lost and the number of plasmid
copies per cell increases continuosly
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At the higher temperature cells grow normally for 2-3 hours, and the products from
genes on the plasmid are over-produced
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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 AND CONS OF CLONING IN A PLASMID VECTOR
PROS
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Plasmid vectors are biologically simpler to use than bacterophage vectors
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The small size of the vector DNA simplifies the purification and analysis of the foreign
DNA
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Large quantities of foreign DNA may be produced easily using a plasmid vector
CONS
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Joining foreign DNA to vector leads to recombinant molecules with disappointing low
transformation efficiencies
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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 λ
(DOUBLE-STRANDED DNA VECTOR)
λ particles consist of a head, which contains λ DNA, and a tail, which is used for cell
adsorption:
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λ GENOME
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Wild-type λ genome consists of linear duplex DNA molecule of 48 513 bp encoding
about 50 genes.
At both ends are complementary 5´ single-stranded regions of 12 nucleotides.
 These cohesive termini are called cos-sites
 When injected into the host cell, the λ genome is rapidly circularized
by the annealing of the cos-sites
Right end cohesive sites
Left end cohesive sites
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TWO PATHWAYS OF BACTERIOPHAGE λ LIFE CYCLE (1)
After λ 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 λ
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 λ 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 λ gene expressed is cl (look next slide) encoding the λ
repressor which binds to two operator regions OL and OR.
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TWO PATHWAYS OF BACTERIOPHAGE λ LIFE CYCLE (2)
Repressors and activators of
transcription determine the
development of phage λ.
LYTIC PATHWAY
LYSOGENIC PATHWAY
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BACTERIOPHAGE λ AS A CLONING VECTOR (1)
Bacteriophage lambda vectors were developed because several observations were made
that suggested that they could complete their life cycles even if foreign DNA was inserted
into a portion of its genome. This suggested that certain regions of the virus were not
essential.
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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
frequently modified or replaced for use as vectors
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The packaging mechanism that inserts DNA into the phage head has a stringent requierment
for DNA size
 Only molecules with cos sites separated by 78-105% of the length
of the wild-type λ genome will be packaged into viable phage
particles – the upper limit on the size of the foreign DNA tht may
be cloned is about 24 kb.
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λ DNA may be packed into infectious phage particles in vitro using mixed extracts of
cells carrying defective λ strain as prophages
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recombinant DNA packaged into λ phage particles may be introduced into E.Coli 10100 times more efficiently thanrecombinant plasmid DNA.
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BACTERIOPHAGE λ AS A CLONING VECTOR (2)
Insertion and/or replacement vectors
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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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Replacement vectors – a non-essential segment of λ DNA called stuffer is removed
and replaced with a foreign DNA:
 can accomodate foreign DNA of size up to 24 kb
 useful for genomic DNA cloning
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BACTERIOPHAGE λ AS A CLONING VECTOR (3)
Insertion and/or replacement vectors
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Insertion or replacement vectors
 Can be used either as insertion or replacement vectors depending
upon the restriction enzyme selected for cloning
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PROS AND CONS OF CLONING IN A PHAGE λ
PROS
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The efficienct of preparing recombinant clones is generally greater in phage than in
plasmids, due to the efficient packaging and delivery mechanism offered in λ
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λ replacement vectors are designed to carry large inserts – up to 24 kb, which
inefficiently in plasmids
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unamplified libraries are easily stored for long periods at 4 °C due to the stability of
the λ phage particles.
CONS
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The large size (24 kb) of the λ arms complicates the restriction analysis of foreign
DNA.
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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)
- Plasmid + cos sites = cosmid
- Hybrid vectors containing one or more bacteriophage λ cohesive ends (cos sites)
 The cos site and associated genetic elements can direct the packaging of
DNA into the λ capsid in an in vitro packaging mix
 When the cosmid and foreign DNA fragments are ligated, the in vitro
packaged recombinant cosmid is an infectious particle that is capable of
injecting its DNA into a host cell
 After injection the cosmid DNA is circularized like phage DNA, but
establishes itself as a plasmid in the recipient cell, and can be selected on
the basis of a vector drugresistance marker
 Can accommodate up to 47 kb of foreign DNA
 Used mainly to prepare genomic DNA libraries
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Major capsid protein
is product of gene E
Endonucleolytic cleavage by product
gene A at cos sites. At each cos site
nicks are introduced 12 bp apart on
opposite strands of the DNA, hence
generating the cohesive termini of
λDNA as it is found in the phage
particle. ATP is requiered for this
process
Simplified scheme showing
packaging of phage-λ DNA
into phage particles.
Assembly proteins, products of genes
W, FII plus completed tails.
____________________________________________________________________
In
vitro
concatemerized
packaging
phage-λ
DNA in a mixed lysate.
Lyse mix
Add ATP and
concatamerized DNA
Mature phage
particles
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FILAMENTOS PHAGE
(SINGLE-STRANDED DNA VECTOR)
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M13, f1 and fd
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Contain single-stranded DNA of about 6.4 kb, which is protected by a coat of 2710
identical protein subunits
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Do not kill its bacterial host:
 About 1000 phage particles are produced per cell generation and
released into the medium
 Formas plaques on a lawn of bacteria which is due to the slower
growth of bacteria wher infected by the phage, not cell lysis
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The single stranded DNA in the virus particle is replicated by a double-stranded
replicative form, called RF, which can be prepared and used for cloning
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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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Lyfe cycle and DNA replication of phage M13
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FILAMENTOUS PHAGE VECTORS
- Unlike phage λ, 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 β-galactosidase α-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.
Why single stranded vectors ?
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Extremeley useful for applications where single-stranded DNA is requiered:
 DNA sequencing by the original dideoxy method
 Several techniques for site-directed mutagenesis
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Not good for long-term propagation of recombinant DNA
 Inserts longer than 1 kb are not stably maintained
 Vectors to fulfill these needs have been developed called phageimides
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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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MODE OF THE REPLICATION DETERMINES
THE LIFESTYLE OF THE PHAGEMID
- 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
- When F+ E.Coli containing the recombinant phageimid is superinfected with f1 phage
called helper phage supplying gene 2 and all the other viral genes requiered for
packaging and secreting DNA molecules, the phage origin of replication is used and
virus particles are secreted
 The vector behaves as a phage allowing to isolate recombinant singlestranded DNA
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BACTERIAL ARTIFICIAL CHROMOSOME (BAC)
- Low-copy number vector containing:
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F-factor (low copy-number origin of replication)
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parA and parB genes – parititioning function maintaining the copy
number of the vector 1-2
- Able to accept large foreign DNA fragments (>300 kb)
- Used extensively for Human Genome Project and other large scale sequencing
projects
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SUMMARY – CLONING VECTORS
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
- Usually produce large quantity of recombinant DNA
- Ideal for foreign fragments up to about 10 kb
- Often used to clone cDNAs
Bacteriophage λ vectors
- High efficiency of preparing recombinant clones due to the efficient packaging and
delivery mechanism
- Can carry large foreign DNA inserts – up to 24 kb
- Often used to prepare genomic DNA libraries
Cosmid vectors
- Can carry very large DNA inserts up to 48 kb in length
Filamentous phage vectors
- Useful for the preparation of single-stranded recombinant DNA
- Ideal for DNA sequencing and mutagenesis techniques
Phagemids
- Can be replicated as plasmids or filamentous phages
- Recombinant DNA can be stably maintained and prepared in single-stranded form
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