MBY 364

THEME 1: Restriction and ligation of DNA molecules

》 Cutting and joining DNA molecules:

• Available endonucleases have little specificity and the chemical methods
produced small fragments of DNA

• Cutting DNA can occur through:

> Sonication: shearing for DNA 300bp in length
Breakage is random
> Stirring in blender: shearing of DNA 8kb in length
> Restriction endonucleases: cut DNA specifically

• Any method that worked = mechanical shearing (blending)

o Long thin threads which constitute duplex DNA molecules are rigid to
be broken by shear forces in solution
o Intense sonication can reduce length of DNA to 300 nucleotide pairs
fragment the DNA too short, might be too random, or reproducible
o Controlled shearing can be done by high-speed stirring in a blender
o Breakage occurs at random with concern to DNA sequences
o Termini consist of short ss regions which may have to be taken into
account in subsequent joining procedures (vector needs to be
complementary to the sticky ends in order to ligate into the vector)

• Phage biologists in the 1960’s elucidated a biochemical basis of the

phenomenon of host restriction modification
o Culmination = purification of restriction endonuclease of E. coli K12
(Meselson and Yuan, 1968)
o K12 cuts unmodified DNA into large discrete fragments, it was
thought that it can recognize a target sequence, but it is random in its
properties.
o Enzyme does bind to recognition sequence, cleavage occurs at
random several kb away from recognition site

• Hemophilus influenzae have an enzyme that recognizes a target sequence in

a duplex DNA and breaks the polynucleotide chain within that sequence,
which results in discrete fragments of defined length and sequence
 • Vectors are capable. Of replicating the DNA independently

• Transformation = introducing foreign DNA into a host organism where it can

replicate and it replicates independently from the host genome
Leads to merging recombinants into the host

• cells become more permeable during heat shock (42oC) ® only calcium
chloride works for E. coli because it disrupts (destabilizes) the membrane ®
creates pores which close when bacteria are grown at 37oC (growth temp) so
as to get DNA into the cell the calcium acts as a carrier molecule because it
has a positive charge so DNA binds to it and introduce it into the cell

》 DNA manipulation enzymes:

• Nucleases:
Enzymes which cut, shorten and degrade DNA
Exo- and endo-nucleases
Exo- removes nucleotides from only the ends if DNA
Endo- cleaves DNA internally between adjacent nucleotides ® cuts
DNA ® shortens fragments and leads to degradation of fragments
Restriction endonucleases ® cuts at specific sequences (restriction
sites) ® more specificity ® reproducible ® outcome becomes
predictable (need to be specific when answering these questions)

• Polymerases:
Copying of nucleic acid template
All require a PRIMER to initiate DNA synthesis
Produces a complimentary DNA stand
DNA Pol 1 has polymerase activity + nuclease activity ® can also
backtrack and repair/edit; associated w/nicks in dsDNA breaks, it
degrades the broken strand and resynthesizes the missing strand
Klenow fragment = the polymerase fragment of DNA Pol 1, fills
single stand nicks and has 5’ ® 3’ polymerase activity and 3’ ® 5’
exonuclease activity (this method of “repair” is NB for when
linkers/adaptors cannot be used to insert DNA fragments into RE
sites, the Klenow fragment can fill the gap of the overhang and
create a blunt end) note, Klenow does not degrade, only fills nicks
Reverse transcriptase – RNA ® cDNA ; is a RNA dependant DNA pol;
creases a RNA/DNA hybrid ® not cloneable so needs to become
dsDNA to be cloned
DNA Pol 1 and Klenow fragment are DNA dependant DNA pols

• Ligases:
Joins DNA molecules together
Repairs discontinuity (breaks/gaps)
Crucial for recombinant DNA formation
 • DNA modifying enzymes:
Modification of DNA through addition or removal of chemical groups
Alkaline phosphatase enzyme = end product is dephosphorylated
Polynucleotide kinase = end product is phosphorylated, uses
dephosphorylated substrates
Terminal deoxynucleotidyl transferase = when supplied with
nucleotides, the enzyme can add the nucleotides to the 3’ end ONLY
of a single or double stranded DNA, to create sticky ends

》 Bacteriological basis of host-controlled restriction and modification of

bacteriophage NDA led to the identification of restriction endonucleases:
• Restriction systems allow bacteria to monitor origin of incoming DNA to
destroy it if it is foreign properties of bacterial host cells is NB

• Restriction endonucleases recognize specific sequences in the incoming DNA

and cleave the DNA into fragments, either site specific or random

• when incoming DNA is bacteriophage genome, the effect is to reduce the

plaques formed on plates

• Plaque assay:
- carried out by overlaying agar plates with soft agar containing bacterial
cells to which bacteriophages have been pre- adsorbed

- During incubation of the agar plates, the bacteria begin to grow

throughout the overlay thereby forming a lawn of cells on the surface of
the agar plate.

- The phage-infected cells lyse, releasing more pages. The presence of the
phage is therefore indicated by a zone of clearing in the bacterial lawn,
known as a plaque

- Plaque assays are used to determine the concentration (titre) of the

phage suspension. The number of plaques in the overlay equals the
number of bacteriophages

• modification and restriction studied by behaviour of phage lambda (lytic

bacteriophage) on two E.coli host strains
o Stock prep of phage lambda is prepared by growth on E. coli strain C,
and this stock is then titrated onto E. coli C and E.coli K, the titres
observed will differ by several orders of magnitude. K being lower
than C

o Phage is said to be restricted by the K strain host phage isolated for

E.coli C was titrated and infected into E.coli K
 o When phage that do result from the infection of strain K are replated
onto K they are no longer restricted; but once they are cycled
through C they are once again restricted when plated onto K

o Endonuclease responsible = restriction endonuclease/restriction

enzyme

12 MARKS: non-heritable change conferred upon the phage by the second host strain, K, that allows it to be
replated on that strain without further restriction. E.coli K phages into E.coli K ® many proliferating phages,
but not in E.coli C ® what happens to the phage depends on the host ® Phage DNA degraded ® low
• Restricted
replication ® low titre phages absorb
® host environment to restrictive
of E.coli hosts and
K32is unsuitable inject
(seen their DNA
as foreign in C)normally
for E.coli C phages
o When
so the phages become degraded andphage is labelled
the titre P, it istitre
lowered. High observed
is due that DNA is degraded
to methylation from thesoon
original
after injection
host, so the phage is protected from restriction enzymes and thus degradation

• Restriction host must protect its own DNA from pot lethal effects of
restriction endonuclease so DNA is modified
o Modification involves methylation of certain bases at a very limited
number of sequences within DNA, which constitute the recognition
site for the endonuclease.

o This explains why phages survive one cycle of growth upon the
restrictive host can reinfect that host efficiently; their DNA has been
replicated in the presence of the modifying methylase and so it
becomes methylated and protected from the restriction system.

o Whenever DNA is transferred from one bacterial strain to another

process can occur

A GOOD CLONING HOST:

- Is integration deficient where DNA cannot
integrate into the host genome
- Must be able to enter the host cell
- Must be restriction enzyme deficient
- Must not be able to perform methylation
Ø EXAMPLE:

E.coli strain Phage titre

Lambda K Lambda B

K 106 10-5 Lambda K and Lambda B prepared

in respective hosts and inserted
B 10-5 106 into E.coli K, B and C.

C 106 106

® 15 MARKS!

® Explain mechanism behind this phenomena:

Same host = high titre ® phage DNA modified through
methylation modified by host specific enzymes

New host = low titre ® different bacteria ® different restriction enzymes

In E.coli C = both high titres ® seems as if DNA is not being cut b/c high
titre present ® restriction enzyme deficient ® DNA irrespective of source
can produce high titres ® good cloning host.

Restricted phages absorb to restrictive hosts and inject their DNA normally.

》 Four types of restriction and modification (R-M) systems have been recognized but
only one is widely used in gene manipulation:
• Type I
First characterised in E. coli K12
Has different subunits: S (x2), M(x2) & R (x1) (hsdS, hsdM & hsdR)
> S = specificity subunits
> M = modification
> R = cutting subunit
Recognition sequences are long with no features like symmetry
Methylation and cutting reactions require ATP, M+2 ions and
S-adenosylmethionine (SAM) as cofactors
Enzyme cuts unmodified DNA at some distance from the recognition
sequence (around 1000bp away) ® VERY UNSPECIFIC
But, because the methylation reaction is performed by the same
enzyme which mediates cleavage, the target DNA may be modified
before it’s cut, This makes type I of little use for gene manipulation,
but presence in E. coli can affect recovery in recombinants
If the target sequence is already methylated then no cutting will occur
so enzyme dissociates; hemi-methylated site cannot be cut; ONLY
unmethylatedsequences can become cut
 these enzymes cut in a random manner ® non-specific cleavage ®
not usefulfor cloning b/c of cutting
NOT useful for gene manipulation

• Type II
Simple protein structure
Has a separate endonuclease and methylase
Requires Mg+2 ions
Cleavage sites is WITHIN the recognition sequence, which is
symmetrical
USED in gene manipulation
These recognition sequences are less frequent in the gene because
they are long (4-8bp)
MOST USED & MOST USEFULL
\ Recognise target sequences of tetra- up to octa-nucleotides
\ Fragment sizes generated e.g.:
§ Tetranucleotide target seq = 44 = 256 bp
§ Hexanucleotide target seq = 46 = 4096 bp
\ They recognise symmetrical target sequences
\ Axis of rotational symmetry & palindromic
\ Cleaves dsDNA yielding blunt ends (flush ends) or sticky ends
(cohesive ends)
ADVANTAGE over Type I & II
\ Restriction and modifications are done by separate enzymes,
so it is possible to cleave DNA in the absence of modification
\ Restriction activities do not require ATAP or any other
cofactor, which make them easier to use
\ Type II enzymes recognise a defined, usually symmetrical,
sequence and cut within it
\ Many also make staggered breaks in the DNA

• Type IIs
Similar to Type II in terms of protein structure and requirements
s = shifter cleavage
recognition sequence is asymmetric and cleavage is up to 20bp away,
so NOT useful for gene manipulation

• Type III
Resembles Type I, except the recognition sequence is symmetrical
Single enzyme with two subunits (M & R)
Require ATP, Mg+2 and SAM
Cleaves 24-26 bp to the 3’ end of recognition sequence
NOT useful in gene manipulation b/c cleaves non-specifically
 RM SYSTEM CAN BE USEFUL BUT CAN UNDERGO MODIFICATION:
- Eco571 system has a single polypeptide which has both modification
and restrictive activities
- Mcr and mrr systems do not fall under this classification
- Homing endonucleases are double stranded DNased derived from
introns and inteins, they have large asymmetric recognition seq and
tolerate some degeneracy within the recognition seq unlike standard
endonucleases

》 Naming of restriction endonucleases provides info about their source

• Smith and Nathans simplified system features:
o Species name of host organism is identified by the first letter of the
genus name and the first two letters of the specific epithet to
generate a 3 letter abbreviation. Abbrev always italic
o Where a particular strain has been the source then this is identified
o When a particular host strain has several different R-M systems, these
are identified by roman numerals

• Homing endonucleases are named similarly except that intron-encoded

endonucleases are given the prefix “I-” and intein endonucleases have the
prefix “PI-”

• Where it is necessary to distinguish between restriction and methylating

activities they are given R and M prefixes

》 Restriction enzymes cut DNA at sites of rotational symmetry and different enzymes
recognize different sequences
• Most type II restriction endonucleases recognize and cleave DNA within seq
of 4-8 nt which have two-fold axis of rotational symmetry

o Such sequences often referred to as palindromes because of their

similarity to words that read the same backwards as forwards

No need to study, theses

5’ overhang = 3’ end
sequences will be given
is longer
in tests and exams
3’ overhang = 5’ end
is longer
 o These DNA fragments can associate by hydrogen bonding between
overlapping 5’ termini, or the fragments can circularize by
intramolecular reaction ® why fragments have sticky ends

》 The G+C content of a DNA molecule affects its susceptibility to different restriction
endonucleases
• Number and size of fragment cut by restriction enzyme depend on frequency
of occurrence of target site in the DNA

• Assuming: 50% GC content and random distribution of bases, four base

recognition site occurs every 44 bp, same for 6 and 8 bp recognition sites
tetranucleotide is 4, hexanucleotide is 6

Ø EXAMPLE:
EcoRI

5’ – G|AATTC – 3’ Step 1 : convert the DNA to dsDNA

3’ – CTTAA|G – 5’

5’ – G AATTC – 3’ Step 2: pull apart the sequences to expose the

3’ – CAATT G – 5’ overhangs

blunt end restriction

enzymes will cut on the axis of
symmetry
 • Isoschizomers = Different enzymes with an identical target sequence, and
cleave the sequence in an identical manner

• Neoschizomers: Different enzymes that recognize identical target sequence,

but cleave the sequence differently

• These sites are important in gene cloning because the sequences of RE’S are
readily cut, if they are methylated, by specific RE’S that have this capability
(some enzymes are indifferent to methylation and will cut either way)

• New restriction sites ® filling in overhangs generated by restricted

endonuclease and ligating them together.

• Blunt-end restriction endonucleases create re-cleavable ligationproducts

> Eg: cleavage with AluI (AG/CT) joined to ones produced by EcoRV
(GAT/ATC); some ligation sites will have seq GATCTor AGATC. Both
can be cleaved by MboI (GATC)

• Methyltransferase isolated from Siroplasma used to modify in vitro

restriction endonuclease target sites which contain the CG sequences
o Some target seq modified with this = resistant toendonuclease
cleavage
Eg: seq CCGG is modified, it will be resistant to HpaII but
sensitive to MspI

》 Methylation can reduce the susceptibility of DNA to cleavage by restriction

endonucleases and the efficiency of DNA transformation
• E. coli = 3 site specific DNA methylases
o Dam genes transfer a methyl group from
S-adenosylmethionine to the N6 position of the adenineresidue in seq
GATC
o Dcm gene modifies internal cytosine residues in seq CCAGGand CCTGG
at the C5 position
o M.EcoKI sites are rare and occur less frequent

• Are of interest:
o Sites for restriction endonucleases may be resistant to cleavage when
isolated from strains expressing the Dcm and Dam methylases
Occurs when a particular base in recognition site of restriction
endonucleases is methylated
 Relevant base may be methylated by one of the methylases if
the methylase recognition site overlas the endonuclease
recognition site.
§ Ex: Dna isolated from Dam+ is completely resistant to
cleavage by MboI, but not Sau3AI,both recognise seq
GATC
§ DNA from Dcm+ strain will be cleaved by BstNI and
resistant to EcoRII, recogn sites = CCATGG

•
Modification state of plasmid DNA can affect freq oftransformation in certain
situations
o Transform efficiency will be reduced when Dam-modplasmid DNA is
introduced into Dam – or Dam- or Dcm-mod DNA is introduced into
other species.
o When DNA is moved from E.coli = use strain lackingDam and Dcm
methylases.
o Difficult to clone DNA with short, direct repetitive seq
o Deletion of repeated seq occurs quickly, even if host strain isdeficient
in recombination
o Deletion mechanism seems to involve Dam methylation, itdoes not
occur in dam mutants
Ø EXAMPLE:

Dam methylation act ® Isoschizomer

- GA*TC MboI |GA*TC : resistant doesn’t digest

SAU3AI |GATC : no effect, can digest

* = methylation

Dcm methylase - methylates 2nd cytosine in sequence ® Neoschizomers

- CC*(A/T)GG EcorRll : |CC*(A/T)GG : resistant

BstNl : CC|(A/T)GG : no effect

Alternative
- Use a host that is methylation deficient and dam and dcm deficient if you
have no isoschizomeres, and neochizomeres are available
 》 Joining DNA molecules : DNA ligase
• When ds breaks occur in DNA

• E.coli requires NAD

• T4 requires ATP

• The nick forms a phosphodiester bond and ligase then closes the gap by
joining the two fragments and it anneals by phosphodiester bonds

• DNA ligase can form phosphodiester bond only when there is exposed 5’
phosphate and 3’ OH

• Understand temp at which ligation occurs, and how we can enhance the
formation of recombinant molecules
Temperature:
\ Optimum temperature for ligases is 37oC to function
\ if we use same RE to cut both the DNA and vector it forms
complimentary sticky ends, WE WOULD NOT ligate at 37oC
because hydrogen bonds between nucleotides will become
unstable and no formation/annealing/ligation can occur
Blunt ends ligation is less \ ligation occurs at a lower temperature than the optimal
efficient than sticky ends temperature (4-15oC) because it is a compromise between
annealing because we get rate of enzyme action and stability of associated DNA ends
\ blunt end ligation and annealing occurs at a higher
fewer recombinants ® we
temperature because there is no unstable sticky ends; high
can increase the insert
temp because it favours faster enzyme activity (blunt ends are
concentration to ensure
incubated with insert and plasmid at 22-25oC)
annealing at more RE sites

Vectors and inserts:

\ we can increase the DNA concentration in tube where there is
an increase in vector association with insert (inter-molecular
interaction)
\ we can also modify the blunt ends by phosphorylase we can
increase annealing ® dephosphorylation can prevent
dimerizing or forming a non-recombinant molecule (figure
4.8), uses alkaline phosphatase to dephosphorylate blunt ends
® insert provides missing 5’PO groups so ligase can joins
insert and plasmid
\ nick from 5’ OH and 3’ OH, is repaired when inserted into
bacterial cell so no problem
 \ T4 is more efficient in blunt ends ligation
\ If we only had E.coli ligase – add something to the ligation
reaction to make the liquid more viscous to increase DNA
interactions (polyethylene glycol (PEG))

• Using Linkers & Adaptors:

o Linkers
> Self-complimentary decameric oligonucleotides
> Contain recognition sites for one or more restriction endonucleases
> Linkers are added ONLY to BLUNT-ENDED fragments
> Is a chemically synthesized piece of DNA that is blunt, contains the
recognition sequence form 1/+ RE’s
> convert insert into something that has compatible ends to the vector
> ligate insert to linker DNA ® digest the new fragment with EcoRI to
create suitable sticky ends for vector
> BUT if we add EcoRI to unknown DNA it could possibly cut in the DNA
insert
> SOLUTION = use an EcoRI methylase enzyme on foreign DNA before
we add the linkers so only ER regions in linkers can be cut (internal cut
sites are protected) ; can also make use of adaptors

Example of the use of linker

DNA to ligate a fragment into
a vector!
 o Adaptors:
> Chemically synthesized DNA containing a blunt end and a preformed
cohesive end
> overcomes problem of internal cut sites in DNA and if we do not have
appropriate methylase enzyme for the fragment
> adaptors also have 1/+ different restriction enzymes on blunt end so
other sticky ends can be formed
> has a BamHI overhang, preformed sticky ends is dephosphorylated
because it prevents self-ligation
> insert DNA has to become phosphorylated (by polynucleotide kinase)
to be cloned into a dephosphorylated vector for ligation to act
> Enable recovery of insert DNA after cloning by restriction with a
different restriction enzyme - thus - adaptor molecule contains other
restriction enzyme sequences

8 MARKS: Clone a DNA fragment obtained by digestion of DNA with EcoRI and HaeIII, into a vector that has
been digested with BaII. Indicate all steps, enzymes and Nucleotide sequences:
- Write down nucleotide recognition sequences
- DRAW PICTURES
Draw out dsDNA sequence
Preform digestion and separate the strands
Draw insert into the vector
- If we had no linkers/adaptors to use:
Do a blunt end ligation
USE KLENOW POL. with dATP and dTTP so that the polymerase can fill in the overhang and
create a blunt end
Draw insert into vector, blunt ends can anneal even if they do not form an RE site
Blunt end ligation is easiest here
we cant use terminal transferase because it will digest the vector and this will lose important
DNA
5 MARKS: Clone and insert DNA that was obtained by restriction enzyme digestion of DNA wit PuvII into a
plasmid vector , which has been digested with HindIII, by making use of an adaptor, following cloning, the
insert DNA must be recovered by digestion of the recombinant DNA with XmaI. Indicate the nucleotide
sequence of your digested sequence of your designed adaptor:
- DNA is cut with different RE than vector was cut
- DRAW IT OUT!
- adaptors have overhangs & blunt ends ® blunt end must be phosphorylated and sticky end must be
dephosphorylated to prevent self-annealing
- insert will be blunt on both sides because of cut with Puvll
- vector will have 5’ overhang
- you HAVE TO USE ADAPTOR, need to have sticky end, and cohesive ends, the blunt ends has Hindlll
sticky end and has a Xmal cut site in blunt end site
- adaptors are designed by us, we fit and mix and match
- adaptors anneal to both ends of the vector if they have to anneal to sticky ends but adaptors only
have on sticky end, thus the adaptor can flip around and upside-down to fit!

》 Cloning DNA by Homopolymer Tailing

• Insert DNA can be cloned into a suitable vector using homopolymer tailing

• The enzyme needed to add homopolymer tails:

® Terminal Transferase = enzyme Continuously adds nucleotides to the 3’ end
of DNA ONLY

• The vector is cut with the appropriate restriction enzyme, followed by

homopolymer tailing, e.g. oligo(dG)

• The insert DNA is tailed with the complimentary base, e.g. oligo(dC)

• These two will anneal by complimentary base pairing, leading to a recombinant

plasmid

• Terminal transferase is only used when NO other option of ligation can be used
and is not common in actual practice
 THEME 2 : Plasmids as cloning vehicles for use in E. coli

》 Basic properties of plasmids:

● Plasmids are replicons which are stably inherited in an extrachromosomal
state. ® never integrated into host genome b/c its difficult to recover
integrated DNA is more difficult & copy number of genome is very low in
comparison to plasmid number
9 Thus:
- The plasmid exhibits genetic homogeneity ®each plasmid must
have the same sequence
- The plasmids are all a constant monomeric unit size
- They can replicate independently from the host genomic DNA
® needs ori

● Most plasmids exist as double-stranded circular DNA molecules →If both

strands of DNA are intact circles the molecules = covalently closed circles or
CCC DNA & If only one strand is intact = open circles or OC DNA.
9 plasmids are found in prokaryotes & most are dispensable but some
contain genes thatconfer a phenotypic result that are essential to
the survival of the bacteria e.g. genes for antibiotic resistance

● CCC DNA is in a supercoiled configuration (Addition of an intercalating agent,

such as ethidium bromide, to supercoiled DNA causes the plasmid to unwind.
If excess ethidium bromide is added, the plasmid will rewind in the opposite
direction)

● Some linear plasmids do exist, and these ends have to be protected against
nucleases:
either there are repeated sequences ending in a terminal DNA hairpin
loop (Borrelia)
the ends are protected by covalent attachment of a protein
(Streptomyces).
6
● Typically the CCC dsDNA size varies in prokaryotes, from 1x10 to 200x106 Da

● Plasmids may also confer a unique phenotypic trait to their host

● Plasmids to which phenotypic traits have not yet been ascribed are called
cryptic plasmids.
● Plasmids can be categorized in one of two major classes, depending on
whether or not they carry a set of transfer genes (tra genes) which promote
bacterial conjugation
Conjugative = carry the tra gene which promotes conjugation

Non-conjugative = absence of tra gene (transfer through chemical

processes)

● Plasmids can also be categorized on the basis of the amount of plasmids that
can be present in a cell
Relaxed plasmid = multiple copies per cell (don’t have tra genes, small
molecular weight)
Stringent plasmid = limited number of copies per cell (high molecular
weight, tra genes present)

● Generally, conjugative plasmids are of relatively high molecular weight and

are present as one to three copies per chromosome

● Non-conjugative plasmids are of low molecular weight and are present as

multiple copies per chromosome (b/c they don't have tra genes)

● Plasmids encode only a few of the proteins required for their own replication
and they are located very close to the oriC site thus only a small region
surrounding the ori site is required for replication. Other parts of the plasmid
can be deleted and foreign sequences can be added to the plasmid and
replication will still occur.
9 useful cloning vector = non-conjugative, relaxed plasmid (high copy
number, lowmolecular weight) lack tra genes

● The host range of a plasmid is determined by its ori region

● Thus the ori is important for:

9 Allowing replication

9 Determining host range e.g. RP4 (conjugative) plasmid has a broad

host range, and will replicate in most Gram-negatives & RSF1010
(non-conjugative) plasmid that also have a broad host range and
canreplicate in Gram-positive and Gram-negatives

● Some plasmids are said to be “promiscuous” in that they are stably

maintained in a diverse range of hosts, this includes RP4 and RSF1010, and
examples of a “restricted” host range plasmid would be for the ColE1 plasmid
that is restricted to enteric bacteria
9 promiscuous plasmids can have an advantage to be inserted into
many bacterial strains (disadvantage = can clone in a undesirable
gene) but we want to use restricted plasmids thatcan be cloned and
expressed in specific bacteria
 ● Promiscuous plasmids offer the potential of readily transferring cloned DNA
molecules into a wide range of genetic backgrounds.

● only small region of plasmid is needed for replication, rest can be removed
and we caninsert foreign DNA into the plasmid without affecting its
replication

》 Plasmid copy number:

● The copy number of a plasmid is determined by regulating the initiation of
plasmid replication and there are two major mechanisms of control of
initiation:
9 regulation by antisense RNA

9 regulation by binding of essential proteins to repeated sequences

called iterons

● Most cloning vectors in current use, use a ColE1 plasmid: (fig 4.4)
9 the primer for DNA replication is a 555- base ribonucleotide molecule
called RNA II, which forms an RNA–DNA hybrid at the replicationorigin
9 RNA II can only act as a primer if it is cleaved by RNase H to leave a free
3′ hydroxyl group. ® RNase H can only cut RNA when it is associated
with DNA ; cut exposes OH for copies to be added to thatend
9 Replication control is mediated by another small (108-base) RNA
molecule called RNA I, which is encoded by the same region of DNA as
RNA II but by the complementary strand
9 RNA I and RNA II are complementary to each other and can hybridize
to from a dsRNA helix

9 if the double helix forms, RNA ll cannot be processed by RNase H so

replication of the plasmid cannot occur more RNA l will be synthesized
when the copy number of the plasmids is high to bind to RNA ll to
regulate copy number
9 In addition to RNA I, a plasmid-encoded protein called Rop (Repressor
of Primer), helps maintain the copy number. This protein, which formsa
dimer, enhances the pairing between RNA I and RNA II so that
processing of the primer can be inhibited even at relatively low
concentrations of RNA I.
9 Deletion of the ROP gene or mutations in RNA I result in increased
copy number
 ● In plasmid PSC101
9 ori region contains three to seven copies of an iteron sequence which
is 17 to 22 bp long. Close to the ori region there is a gene, called repA,
which encodes the RepA protein.
9 RepA proteins are the only plasmid-encoded proteins required for
replication, which then binds to the iterons and initiates DNA
synthesis
9 And in this plasmid, copy number control is exerted in two ways:
o RepA proteins repress their own synthesis by binding to its
own promoter and blocking transcription of its own gene → if
↑ copy number then RepA is repressed
o RepA can also cause handcuffing between two iterons
preventing replication initiation
o Replication of iteron plasmids will depends on both [RepA]
plasmids themselves

》 Stable maintenance of plasmids in cells:

● The loss of plasmids due to defective partitioning (segregation when the cell
divides) = segregative instability

● Naturally occurring plasmids are stably maintained because they contain a

partitioning function, par, which ensures that they are stably maintained at
each cell division. Such par regions are essential for stability of low-copy
number plasmids

● Partition-defective mutants of pSC101 and similar mutants of unrelated

plasmids are stabilized in Escherichia coli by topA mutations, which increase
negative DNA supercoiling (DNA gyrase inhibitors and mutations in DNA
gyrase increase the rate of loss of par-defective pSC101 derivatives)

● Plasmid instability may also arise from the formation of multimeric plasmids
that have the same number of plasmid origins but fewer plasmid molecules

● Multimeric forms are not seen in ColEl which naturally resolves multimers
because it contains a highly recombinant site (cer), which if it occurs more
than once in a plasmid, as in a multimer, the host cell’s Xer protein will
promoter recombination between these sites and resolve the multimer

● Plasmid incompatibility = the inability of two different plasmids to coexist in

the same cell in the absence of selection pressure.

● Groups of plasmids which are mutually incompatible are considered to

belong to the same incompatibility (Inc) group
 ● Plasmids will be incompatible if they have the same mechanism of replication
control or alternatively, they will be incompatible if they share the same par
region

》 Purification of plasmid DNA:

● A prerequisite for cloning is the purification of the plasmid DNA so the DNA
has to be extracted from the cells

● Extraction of plasmid DNA is done first, by cell lysis which is the most difficult
step; ideally each cell is just sufficiently broken to permit the plasmid DNA to
escape without too much contaminating chromosomal DNA

● Provided the lysis is done gently, most of the chromosomal DNA released will
be of high molecular weight and can be removed, along with cell debris, by
high-speed centrifugation to yield a cleared lysate

● There are 2 methods to isolate pure plasmid DNA from cleared lysate
discussed here:
Method 1: Alkaline lysis (method of practical)
o Most commonly used method
o Method makes use of the narrow pH range (12-12.5) within
which denaturation of linear DNA, BUT NOT CCC DNA, occurs
\ Treat cells with lysozyme to weaken the cell walls
\ Treat cells with NaOH (sodium hydroxide) and SDS
(sodium dodecyl sulfate to lyse the cells →
chromosomal DNA now denatured (high pH) (lysis
bacterial cell walls as well, the two strands between
CCC DNA do not separate, still inter-linked)
\ Add acidic sodium acetate to neutralize the solution,
causing the chromosomal DNA to renature and
aggregate (mismatch between DNA, partial
complementarity) into an insoluble network, as well
asproteins and high molecular weight RNA to
precipitate
→ the CCC plasmid DNA molecules will remain in a
native state and in solution, while the contaminating
macromolecules co-precipitate (low pH)
\ Precipitate can be removed by centrifugation and
theplasmid concentrated by ethanol precipitation
\ The plasmid DNA will be found in the supernatant
 Method 2: CsCI isopycnic centrifugation
1. Centrifugation of cleared lysate in a CsCl solution
containing ethidium bromide (EtBr)
2. The EtBr intercalates between the DNA base pairs and
causes the DNA to unwind
3. CCC DNA has no free ends so can only unwind to a
limited extent, limiting the amount of EtBr the can bind
→ density of the DNA–EtBr complex decreases as more
EtBr is bound, so CCC DNA has a higher density at
saturating concentrations of EtBr
4. Linear DNA (e.g. chromosomal DNA) binds more EtBr
because it has exposed ends and can also unwind more
→ low density
5. Thus CCC DNA and linear DNA can be separated by
centrifugation based on density

》 Desirable properties of cloning vectors:

● An ideal cloning vehicle would have the following three properties:
- Low molecular weight
> Easy to handle (readily isolated from host cells)

> Present in high copy numbers

> Lower probability of multiple restriction endonuclease

recognition sites
- Confer readily selectable phenotypic traits on the host cell
- Unique restriction endonuclease recognition sites (preferably in
genes with a readily scorable phenotype)

● One of the first steps in cloning is to cut the vector DNA and the DNA to be
inserted with either the same endonuclease or ones producing the same ends

● It is advantageous if insertion of foreign DNA at endonuclease-sensitive sites

inactivates a gene whose phenotype is readily scorable, for in this way it is
possible to distinguish chimeras from cleaved plasmid molecules which have
self-annealed.

● pBR322 plasmid
9 Plasmid pBR322 contains the ApR (Ampicillin resistant) and TcR

(Tetracycline resistant) genes of RSF2124 and pSC101, respectively,

combined with replication elements of pMB1, a Col E1-like plasmid
9 Contains an ori, consists of 4363 bp, fully sequenced
9 The DNA sequence of pBR322 characterizes it in terms of restriction
sites, such that the length of every fragment can be calculated
9 Has over 40 unique restriction sites: 11 in Tet resistance gene (+2 in
promoter) , and 6 in Amp resistant gene
 9 result in insertional inactivation of either the ApR or the TcR markers
9 E. coli cells transformed with plasmids with inserts in the TcR gene can
be distinguished from those cells transformed with recircularized
vector → former are ApR and tetracycline sensitive (TcS), whereas the
latter are both ApR and TcR
9 transformants are selected on the basis of their Ap resistance and
then replica-plated onto Tc-containing media to identify those that are
TcS (Tetracycline sensitive)
9 Detection is based upon the ability of the β-lactamase produced by ApR
cells to convert penicillin to penicilloic acid, which in turn binds iodine,
which forms clearing zones. Transformants are selected on richmedium
containing soluble starch and Tc. When colonized plates are flooded
with an indicator solution of iodine and penicillin, β-lactamase-
producing (ApR) colonies clear the indicator solution whereas ampicillin
sensitive (ApS) colonies do not.

Ø EXAMPLE : the use of plasmid pBR322 as a vector: isolation of DNA

fragments which carry promoters

o Cloning into the HindIII site of pBR322 generally results in loss of

tetracycline resistance because he HindIII site lies within the
promoter

o whether or not insertional inactivation occurs depends on whether

the cloned DNA carries a promoter-like sequence ableto initiate
transcription of the TcR gene

o Four structural domains can be recognized within E. coli promoters

- position 1, the purine initiation nucleotide from whichRNA

synthesis begins

- position -6 to -12, the Pribnow box

- the region around base pair −35

- the sequence between base pairs −12 and −35

- the Hindlll site lies within the Pribnow box, but the box is
recreated on insertion of the foreign DNA, thus when
insertional inactivation occurs, it must be in the region from
-13 to -40 which is modified
 ● pUC vectors
9 each new vector was constructed in a series of steps analogous to
those used in the generation of pBR322 itself, but construction of
vector was improved by the use of polylinkers or multiple cloning sites
(MSCs), as exemplified by pUC vectors
9 MSC = short DNA sequence, 2.8 kb in pUC19, carrying sites for many
different restriction endonucleases
9 MSCs increase the potential number of cloning strategies available by
extending the range of enzymes that can be used to generate a
restriction fragment for cloning
9 pUC vectors contain an Amp resistant gene, an ori, consists of
2700 bpand is fully sequenced, and contains MSCs within
which a lac Z’ gene is
9 The pUC vectors also incorporate a DNA sequence that
permits rapid visual detection of an insert.
o The MCS is inserted into the lacZ′ sequence, which encodes
the promoter and the α-peptide of β-galactosidase.
o Insertion of MSC does not affect the ability of α-peptide to
mediate complementation, but cloning of DNA into the MSC
does
o recombinants can be detected by blue/white screening on
growth medium containing Xgal
o The usual site for insertion of the MCS is between the initiator
ATG codon and codon 7, a region that encodes a functionally
non-essential.part.of.the.α-complement

Why put it into a vector?

- To be able to insert into the host
- To replicate the gene of interest
- To ensure an origin of replication is present
- Plasmid = replicons ; stably inherited from one cell to another

If DNA is not in a vector, it is difficult to recover the integrated DNA, the fragment copies might be very low
and if it is independent of the host genome it can easily purified and extracted from the DNA