Contents

Vectors: introduction .................................................................................................................... 1

DNA manipulation: enzymes ......................................................................................................... 4
Introduction of DNA into living cells ............................................................................................. 8
Tranformation ........................................................................................................................... 8
Conjugation ............................................................................................................................. 11
Introduction of phage DNA ..................................................................................................... 12
Introduction of DNA in non-bacterial cells.............................................................................. 14
Cloning vectors ............................................................................................................................ 16
For bacteria ............................................................................................................................. 16
Cloning vectors based on the M13 phage ........................................................................... 16
Cloning vectors based on the λ phage ................................................................................ 18
For yeast .................................................................................................................................. 21
For plants................................................................................................................................. 23
For insects ............................................................................................................................... 25
Polymerase Chain Reaction ......................................................................................................... 26
How to obtain a clone of a specific gene .................................................................................... 28
Identification of a clone from a gene library ........................................................................... 28
Southern hybridization ........................................................................................................ 29
Mutagenesis and interference .................................................................................................... 31
In bacteria ............................................................................................................................... 31
Transposon mutagenesis..................................................................................................... 31
Deletion of genes ................................................................................................................ 32
Marked mutants .................................................................................................................. 35
Insertional inactivation........................................................................................................ 36
In yeast .................................................................................................................................... 36
siRNA interference .................................................................................................................. 38
Site-directed mutagenesis....................................................................................................... 39
Overview ................................................................................................................................. 40
DNA sequencing .......................................................................................................................... 41
Chain termination method ...................................................................................................... 41
 Chemical degradation method................................................................................................ 42
Sequencing genomes .............................................................................................................. 43
Studying gene expression and function ...................................................................................... 44
Northern hybridization............................................................................................................ 45
Nuclease protection assay ...................................................................................................... 45
Quantitative reverse transcription PCR .................................................................................. 45
Reporter genes ........................................................................................................................ 48
DNA microarrays ..................................................................................................................... 48
cDNA microarrays................................................................................................................ 49
Oligonucleotide arrays ........................................................................................................ 49
Expression of recombinant proteins ........................................................................................... 51
Overexpression in bacteria ..................................................................................................... 51
Expression in eukaryotes......................................................................................................... 54
Some examples ....................................................................................................................... 55
Western blot ............................................................................................................................... 56
Proteomics .................................................................................................................................. 57
Interactomics............................................................................................................................... 58
Genetic approaches: Two-hybrid systems .............................................................................. 58
Biochemical approaches ......................................................................................................... 59
In vitro ................................................................................................................................. 59
In vivo: using tags ................................................................................................................ 61
Molecular typing methods .......................................................................................................... 62
Genome analysis by pulsed-field gel electrophoresis (PFGE) ................................................. 62
Ribotyping ............................................................................................................................... 63
Randomly amplified polymorphic DNA fingerprints (RAPD) ................................................... 64
Multilocus sequence typing (MLST) ........................................................................................ 64
Epigenetics .................................................................................................................................. 65
Cystic Fibrosis .............................................................................................................................. 69
Molecular imaging....................................................................................................................... 71
SPECT and PET probes ............................................................................................................. 72
In vivo imaging of gene expression ......................................................................................... 74
Vectors: introduction
Vectors can be plasmid or bacteriophage chromosomes. Vector’s characteristics:
 Must have an origin of replication: they can multiply independently
 Must be small: large molecules tend to break

Plasmids
Plasmids are usually circular molecules (the linear ones aren’t used for cloning). They’re not
essential for bacterial survival in normal conditions. They‘re used in extreme conditions, like
the presence of antibiotics in the environment: that’s why some natural plasmids have
resistance genes.
 Size and copy number  Large plasmids are usually stringent: only one copy per cell.
This type of plasmids is not useful.
 Integration  Plasmids are non-
integrative (the majority) or
episomes. The episomes can be
maintained or excised.
 Compatibility  To be in the same
cell 2 plasmids have to be from
different compatibility groups,
otherwise one of them will be lost.
 Conjugation  A conjugative
plasmid has the tra and mob
genes, being able to mobilize itself.
However, a non-conjugative
plasmid that only has the mob
gene ca be co-transferred with a conjugative plasmid.
 Classification
o Fertility plasmids  They have the tra genes, which enables the cells to
conjugate
o Resistance plasmids
o Col plasmids  They produce toxins (colicines) that kill other bacteria from
the same species in situations of nutrient shortage
o Degradative plasmids  They have genes involved in the metabolism of
xenobiotics, sugars, etc
o Virulence plasmids

Bacteriophages
Virus that infect bacteria and have a simple structure: one DNA molecule + proteic protective
coat/capsid.
λ phage: double stranded ; M13 phage: single stranded
Lytic cycle  involves the destruction of the host ; Lysogenic cycle  doesn’t involve the
destruction of the host

1
λ phage
Lysogenic cycle  After injection, the DNA
circularizes and integrates the host chromosome.
The integrated form of the phage DNA is called the
prophage, and the bacterium that carries it the
lysogen. If the bacterium is in stress the prophage is
excised and circularizes, entering the lytic cycle to
infect new hosts (survival mechanism).

The λ phage has cos sites. These are single stranded

sites found in the linear form of the phage. They
enable:
 The circularization in the cell
 The enzymatic cut necessary during the
replication that divides the newly formed
linear molecules.
The replication is done in a rolling cycle, producing a
lot of DNA copies at once (a catenane). The cos sites
are recognition sites for endonucleases, allowing
the cleavage of the catenane in different DNA
molecules.

2
 M13 phage
Cycle  The ss DNA is injected through the bacterium’s pilus. Then the cycle can go in two
ways:

The DNA is replicated by the same process used in the

A second strand is synthetized, producing a
dsDNA (replicative form). These replicate,
giving rise to new ds molecules. Through a
rolling cycle replication new ss forms are
produced and mature phage are extruded.
bacterium. New M13 phages are continuously extruded
without killing the host, but slowing its division due to the
energy expenditure.

This means that the M13 phage can be both in the ss and ds form within the cell. It’s in the ss
form in the extruded phages.

How to isolate plasmids from bacteria?

Pellet: debris and

chromossomes
Cell SDS Alcohol
NaOH Acid Centrifuge
culture Lysozime (precipitation)
Supernatant:
plasmids

Lyzosime + SDS  disrupts peptidoglycan and produces cell extracts

NaOH  pH increases to 12 and DNA denatures

3
Acid  Quick pH decrease to 4: the larger DNA molecules (chromossomes) don’t have time to
renature and precipitate. The small plasmids renature and stay in solution.
Phenol and chloroform can also be used to do the cleaning step.

DNA manipulation: enzymes

To produce recombinant DNA molecules, the vector and the DNA fragment may have to be
modified:
- shortened
- lengthened
- copied into new DNA molecules
- modified by the addition or removal of specific chemical groups

DNA manipulative enzymes:

 DNA modifying enzymes
o Nucleases
o Polymerases
o Enzymes that act on termini
 Restriction endonucleases
 Ligases

Nucleases
Exonucleases remove one N at a time from the end of the molecule.
Endonucleases break phosphodiester bonds within the molecule.
These enzymes cut in a random way and can be specific for ds, ss or both.
 The DNAseI cuts both forms and is used in RNA isolation.

Polymerases
 Polymerase I: involved in
DNA repair (small breaks).
This enzymes attaches to
small ss regions and
synthesizes a new strand,
filling the gap and replacing
the existing Ns after the
gap. This means that it has a
dual activity: polymerization
and degradation.
 The Klenow fragment is the fragment of the protein that has only the
polymerase activity. It is used to adapt the target DNA to a plasmid without
replacing the existing nucleotides. A ligase has to be used after to bind the
new Ns to the old ones.

4
  Taq polymerase: is thermostable but doesn’t have a proofreading (3´-5´exonuclease)
capacity, having a low fidelity (error rate: 1/1000). So other polymerases with this
capacity are commonly used (ex: Pfu).
 Reverse transcriptase: used in quantitative real-time PCR, micro-arrays and cDNA
libraries

Enzymes that act on termini

 Alkaline phosphatase: it takes out the phosphate groups of DNA molecules.
It used to prevent the vector DNA from recircularization after it’s digested with a restriction
enzyme. However, it also prevents the ligases from binding the target DNA to the vector. This
means that after the insertion there’ll be a nick in each strand in the region s where there’re
OH groups in both strands

.
 Terminal deoxynucleotidyl transferase (TdT): adds random dNTPS to the 3’ terminus
It is used to create homopolymer tails, to insert fragments in a vector (sticky ends).

Restriction endonucleases

Host-controlled restriction
These enzymes cleave exogenous DNA, preventing phage infection. They don’t bind to the
bacterial DNA because the recognition sites are methylated.

5
E.coli K has the system and E.coli C doesn’t.
The phages produced by E.coli K (λ.K) are
modified and when they reinfect E.coli k they
aren’t recognized.
The λ.C aren´t modified so they’re susceptible to
endonuclease digestion when they infect E.coli K,
having a low efficiency of plating.
Both λ.C and λ.K can efficiently infect E.coli C
because it doesn’t have these endonucleases.

There are 4 types of restriction and modification

systems. Only type II is used because the others cleave far Efficiency of Plating (EOP):
away from the recognition site. Relative number of plaques that a
Recognition sites are small (4/6 Ns) palindromic regions; phage stock is capable of
some of them are degenerated. producing

Nomenclature:

Some enzymes make the cut in the same position for both strands, creating blunt ends and
other make this cut with a small distance, creating sticky ends.

Restriction endonucleases with different recognition sites may produce the same sticky ends.
We can use BamHI for the
fragment and BglII for the
vector. However, if after
cloning we want to excise
the fragment we can’t use
neither of them because
the recognition site is
altered: one extremity has
the BamHI part and the
other has the BglII part. In
that case, we could use
Sau3A.

6
Ligases
In the cell these enzymes are used in replication, reparation and recombination.

They can be used to join blunt ends. However, this isn’t very efficient because the association
of the 2 DNA molecules is by chance and only then can the ligase bind these molecules. To
increase the efficiency high DNA concentration must be used and a temperature decrease is
necessary to inhibit DNA motion and increase the chances of association. A temperature
gradient can be used to assure that in some period of time the ligases will have their optimal
temperature.
Ligation of complementary sticky ends due to the base-paring between the 2 molecules gives
rise to a stable structure.

Convertion of blunt ends into sticky ends

Linkers  They’re short pieces of ds DNA.
1st the linkers are attached to the fragment and vector. Since they’re small and are used in
high concentration this reactions is efficient.
Then a restriction endonuclease specific to the linker
sequence is used in the fragment and vector,
creating complementary sticky ends.

Adaptors  They’re short oligonucleotides with one

palindromic sticky end that has an OH in both
strands. They’re ligated to the fragment and the
modified 5’ OH terminus prevents ligations between
adaptors.
After the ligation a polynucleotide kinase is used to
give a phosphate group to the 5’ end. The vector has
to dephosphorylated with alkaline phosphatase to
enable its ligation to the fragment.
We can also don’t dephosphorylate the vector and it
will bind to the OH ends of the fragment. But this
may create recircularization problem.
In this approach there’s no need to use restriction endonucleases.

Homopolymer tailing  Using TdT. The N used in the vector must be complementary to the N
used in the fragment. Since we can’t control the size of the
tail, after the ligation there will be ss regions in the plasmid.
The klenow fragment can be used to repair this.

7
Introduction of DNA into living cells
(the 1st part of the chapter only refers to bacterial cloning)

Cloning can be used to:

 Obtain large amounts of DNA
 Purify DNA  Eliminate circularized vectors without the target gene, vectors with the
wrong fragment, unligated vectors and unligated fragments
Unligated vectors and fragments are rarely replicated because the host enzymes
degrade them. The cells with vectors without the target gene can replicate them but
we can manage to eliminate these colonies with selection markers.

Tranformation
Uptake of plasmids from the medium
1. Adhesion of the exogenous DNA to the DNA binding protein in the plasma membrane
2. Endonuclease in the membrane cuts one strand
3. Specific competence protein

8
If there’s a region of homology between the plasmid and the chromosome the plasmid will be
integrated.

Artificial transformation
Not all cells have the natural ability to take up DNA from the environment (E. coli isn’t
naturally competent). A chemical or physical treatment can enhance this ability to make them
competent. The protocol used is:
1. Suspend the bacterial pellet (log-phase) in CaCl2 solution and store at -80ºC
improves DNA binding and affects the cell wall
2. Add plasmid
3. Heat shock (42ºC)  improves DNA uptake
The use of metal ions can also help the process.

It is also necessary to use an E.coli strain that is restriction- deficient, otherwise this system will
degrade the exogenous DNA.

Electrotransformation
The electric pulse creates transient holes in the cell membrane.
Factors affecting the efficiency:
 Temperature
 Electric field parameters
 DNA form
 Host factors: genetic background, growth conditions

9
 •Versatility
Advantages •Eficiency: even with bigger DNA molecules
•Low amounts of DNA needed

•The cells might be damaged with wrong pulses

Disadvantages •It may create ion imbalance and cell death (due to
unspecific ion transport across the membrane)

Selection of transformants
Transformation of competent cells is inefficient: only 0.01% of the DNA molecules are up
taken. This means that we need to select only the cells that have been transformed. Using
selective mediums with antibiotics we can eliminate the cells that don’t have the plasmids
(which contain antibiotic resistance genes). The transformed bacteria don’t immediately start
replicating the plasmids and expressing these genes: that’s why they must be placed in liquid
medium for a while (1 h) and then plated in selective medium.

Identification of recombinants
Between the transformants are bacteria with vectors:
 With no inserted fragment
 With the right inserted fragment
 With the wrong inserted fragment

To select just the ones which have an inserted fragment we exploit insertional inactivation.
Resistance
markers 
When we use
BamHI to cut
the pBR322
plasmid and
insert the
fragment we
are
disrupting the tetracycline resistance
genes. This means that a transformed
and recombinant bacteria will be
resistance to ampicillin but
susceptible to tetracycline.

To select these recombinants we

replate some colonies from the
ampicillin plate in agar plates
containing tetracycline. By identifying
which colonies in the original

10
ampicillin plate don’t grow in tetracycline plates we get the recombinants.

LacZ  A normal E.coli contains

a lacZ gene enconding for Beta-
galatosidase that lacks a
segment. The pUC8 plasmid has
this segment and the union of
the products of these 2 genes
gives rise to active enzymes.
These enzymes are capable of
breaking down a lactose analog,
X-gal, producing a blue
substance.
So if we used an X-gal-
containing medium:
a non-recombinant bacterium
has active enzymes and
produces blue colonies;
recombinant bacteria doesn’t
have active enzymes due to
insertional inactivation and
produces white colonies.

Conjugation
Conjugative plasmids have the tra genes.
These are responsible for the nicking and
unwinding of the ds plasmid. One strand
stays in the donor cells and its
complementary is synthesized. The other
strand enters the recipient cell through the
pilus and its complementary is synthesized
there. These genes are also responsible for
pilus formation.
The mob genes are also necessary in the
donor cell.

11
 In triparental
conjugation a donor
cell transfers its
conjugative plasmid to
a 1st recipient. This
recipient then can
transfer its non-
conjugative plasmid
(mob+ tra-) to a 2nd
donor cell

Introduction of phage DNA

Transfection
The phage DNA is mixed with competent E.coli cells and DNA uptake is induced by heat shock.
This process isn’t very efficient, when compared with infection with mature phages.

In vitro packaging
λ phage:
This process requires proteins encoded
in the λ genome. To get these, defective
λ strains are used to infect E.coli cells.
 Single strain system:
These don’t have the cos sites so the
catenane can’t be cleaved by the
endonuclease and the phage can’t
replicate. However, phage proteins and
synthesized and can be purified.
 Two-strain system:
Strains mutant for the gene D and gene
E are used. These genes code proteins
for the phage coat and so neither of
these strains can replicate but proteins
are synthetized. By using of mix of the
proteins from the strains we get all the
protein necessary for assembly for λ
phage.

12
Then we just have to mix the packaging proteins and the phage DNA (in the catenane form),
the assembly occurs automatically.

Selection of transformants
Infected cells in agar plates are visualized as plaques. With
lytic phages, like λ , these plaques are clearing zone filled
with lised cells and phages. With lisogenic phages, like M13,
the plaques have slow growing bacteria.

Identification of recombinant phages

 LacZ  This can also be done using

insertional inaction of the lacZ gene, carried in the
phage vector.
 cI gene in λ vector  Insertional
inactivation can also be used in the cI gene, present
in some λ vectors. This gene is responsible for a
turbid plaque appearance.
 Spi phenotype  If an E.coli cell has the P2
prophage, a normal λ vector can’t infect it and is
Spi+. Insertion of a gene in some λ vectors induces
its change to a Spi- phenotype, enabling it to infect
the E.coli cells with the P2 prophage. This way, only
the recombinants will form plaques.
 λ genome size  If a DNA molecule has
less than 37 kb the λ packaging system doesn’t
work. If the λ vector is designed in a way that it just
has 37 Kbs if the gene of interest is inserted, then only recombinant DNA molecules
will be packed.

13
Introduction of DNA in non-bacterial cells

Yeast
1. Grow S.cerevisiae to mid-log phase
2. Treat with lithium/cesium acetate  Lithium ions disrupt the cell wall and
neutralize the DNA charge
3. Add plasmid DNA, carrier DNA, histamine, PEG and TE/CationMIx
 Carrier DNA (ss): in very high concentrations; helps the plasmid DNA
entering the cell and protects it from endonucleases
 PEG (polyethylene glycol): helps the DNA localization closer to the
membrane
4. Heat shock and plating

Electroporation, biolistic and spheroplast methods can also be used.

Animal cells
 Physical transfection  microinjection (also used in plants), particle bombardment
(biolistic method), ultrasound, electroporation
 Chemical-mediated transfection  Calcium phosphate is used to precipitate DNA in
the cell membrane; the precipitates are taken up by endocytosis. It’s suitable for cells
in monolayers, not in clumps.

 Fusion with DNA-containing lysosomes  The DNA is in the lysosomes and they fuse
with the plasma membrane; the DNA goes to the nucleus.
 DNA packed inside a bacteria
 Transduction: virus infection

Plant cells

Bacterial gene delivery: Agrobacterium mediated

transformation.
Agrobacterium tumefaciens naturally infects the stems of
plants. It injects the TI plasmid and a fragment of that
plasmid, the T-DNA, is inserted in the plant genome. This T-
DNA carries genes for tumor formation (crown gall) and
synthesis of opines, amino acid derivatives that the
bacteria can use as a C and N source.

14
How can bacterial genes and promoter work in plant cells? These genes have plant promoters
due to evolution; they don’t even work in Agrobacterium.

If a recombinant A. tumefaciens is introduced

in a plant stem, only the cells in the crown gall
will be recombinant.
To create a recombinant plant, we use plant
cells or protoplast suspensions and transform
them with A. tumefaciens containing a
disarmed TI-plasmid, which doesn’t have the T-
DNA with the oncogenic genes, only the
repeated sequences flanking it, that are
necessary for integration.
The cells are then plated in a selective medium
to select the transformants and these will give
rise to a whole
plant.

Other strategies
 Chemical direct DNA transfer
1. The cell wall is degraded: protoplast formation
2. DNA is precipitated in the protoplasts
3. The transformed cells have to be regenerated before putting them in a medium,
otherwise they won’t be able to grow
4. Plating and selection
 Viral transduction
 Physical direct DNA transfer
Supercoiled DNA plasmids are used. They enter the nucleus (the exact mechanism is unknown)
and integrate the genome by non-homologous recombination.
o Particle bombardment (biolistics)
o Electroporation
o Microinjection

15
Cloning vectors

For bacteria
We want a vector that allows an easy
purification, high transformation efficiency,
selectable markers and ability to clone up to 8
kb.

One of the 1st vectors was pBR322. This plasmid

is small, has 2 resistance genes, unique
restriction sites, 15 copies/cell and is
conjugative. It was made from 3 plasmids: R1,
R6-5 and pMB1.

pUC plasmids
These plasmids have 500-700 copies/cell and a
lacZ’ gene, which means that identification of
recombinants can be done in just one step.
It also has a polylinker/multiple cloning site: short
region of DNA that contains multiple recognition sites.
The presence of these multiple cloning sites widens the
range of enzymes that can be used to cut the target
gene.
The polylinker is located in the lacZ’ gene but doesn’t
affect its expression (unless a fragment is inserted)
because it’s in a region that encodes a non-essential
part of the peptide. It has been reported that
insertional activation only occurs when the fragment is
inserted in a certain region.
This selection marker only works in hosts that have the
lacZ gene (some E.coli strains), since the plasmid only
encodes part of the protein.

Cloning vectors based on the M13 phage

M13: 6407 bp
Since the vector is in the ss form in the cell, this vector offers the possibility of obtaining ss
DNA, important for sequencing and site-directed mutagenesis.
Since the genome is very dense, there was little space to delete DNA so that new fragments
could be inserted.
The region chosen was near
the ori, where a lacZ’ gene is
located with a polylinker
region in this locus.

16
When the vector is treated with restriction endonucleases and religation is done there’re 3
possible outcomes:
 The fragment inserts  lacZ’ disrupted
 Nothing inserts (self ligation)  lacZ’ reformed
 Polylinker reinserts  lacZ’ reformed. This can happen because when the
endonuclease is added a part or all of the polylinker is excised.

The insertion of the vector slows down the growth due to the energy expenditure necessary to
replicate the ss form and extrude the phage, which is going to infect the surrounding bacteria.
So when we plate the mixture we can see plaques that correspond to the slow growth and not
to lysis.
With fragments > 1.5 kb the cloning efficiency drops.

Phagemids
(image in the right)
Hybrids: an M13 fragment was cloned in a plasmid,
allowing the production of ssDNA without having the
fragment size restriction that happens when using the
M13 phage. We can clone fragments up to 10 kb.

The fragment cloned has the signal sequence responsible

for the conversion of dsDNA into ssDNA. We have to
infect in parallel with M13 phage: it functions has a
helper phage that synthesizes all the proteins and
replicative enzymes so that the ssDNA can be assemble
in a phage particle.

Selection is done by insertional inactivation of the lacZ

gene: the transformants are the white plaques.

17
Cloning vectors based on the λ phage
These phages had 2 problems that needed solving before use:
 We could only clone up to 3 kb due to the capsid head size
 There were multiple restriction sites, which means that when treated with
endonucleases the product was a lots of fragments

To solve the 1st problem they took out the region of the genome that had the genes involved in
insertion in the host genome. This meant more space for cloning (up to 18kb) and the DNA
wouldn’t integrate the genome (before this approach these vectors weren’t useful).

To remove the recognition sites natural selection is used:

E.coli cells are infected with a λ phage and plated. Since E.coli has host restricted modification
systems, the phages that have recognition sites will die (most of them). So in this round very
few plaques will be seen: resulting from bacteria lysed due to infection with mutant λ phages
that have less recognition sites. After many rounds the plaques will be constituted of bacteria
lysed due to phages with no
recognition sites.

Cloning the λ phage can be done in 2

ways:
 Cloning the circular vector
(more difficult)
 In vitro packaging
The vector is treated with
endonucleases, being opened at the
recognition site and at the cos site. This
generates 2 λ arms.
The fragment of interest is added
(flanked by 2 recognition sites).
A ligation step is done, leaving a
catenane made up of multiple linear
vectors.
An in vitro packaging mix is added: it
has an endonuclease, that cuts at the
cos sites, and the components of the capsid, generating recombinant λ phages.
The selection of recombinants is done by size: only if the gene inserts, the λ will have the right
size to be packed.

Cosmids
Hybrids: the region of cos site of a λ phage was cloned in a plasmid.
This is because in vitro packaging works in any molecule that has cos
sites separated by 37-52 kb of DNA.

18
Small: 4-6 kb; allow cloning of fragments up to 47 kb (the big advantage comparing to λ
phages).
They need a selectable marker, like ampicillin, and an ori, since they don’t have the λ genes
involved in replication.
They’re an efficient way to introduce DNA in the cell and behave like a plasmid in the cell. This
means that the resulting transformants aren’t
plaques, but infected colonies.

The use of these vectors allows cloning of big

fragments and the use of in vitro packaging.
The cosmid is digested with an endonuclease,
creating a linear cosmid. The fragment (flanked by
recognition sites) is added and ligation occurs,
creating a catenane. The in vitro packaging mix is
added and recombinant λ phages are created. After
infection the cells contain a circular cosmid.

The selection is by size: the in vitro packaging mix

only cut the catenane if the insertion of the
fragment occurs.

Genomic libraries

These high capacity vectors referenced are used for genomic libraries in prokaryotes. Since
eukaryotes have big genomes with a lot of intergenic regions, gene libraries are more common
for these organisms.

Why are genomic libraries useful?

Example  If we have a bacterium that shows a mutant phenotype and we want to know
which is gene is involved. We transform the mutant with clones from the libraries and observe
the absence/presence of complementation. If we see complementation, we sequence the
clone.
If we have 2000 clones we start by transforming with the first 1000 clones (all together). If we
see complementation there, we do it with the first 500,etc…

19
Vector pUC plasmids M13 phage Phagemids λ phage Cosmids
Characteristics  Polylinker in the  Polylinker in lacZ’  M13 fragment  Cos  The cos site of λ
lacZ’  Ori cloned in a plasmid  Unique restriction was cloned in a
 Amp  Only fragments up (allows replication) site plasmid (allows
 Ori to 1.5 Kb  Amp  Fragments up to replication)
 500-700  lacZ’ with 20 Kb  Ori
copies/cell polylinker  Amp
 Identification in a  Fragments up to  Fragments up to
single step 10 KB (advantage 47 Kb
to M13 phage)
Cloning process Restriction enzymes and Cloning in the polylinker Cloning with linear DNA: Cloning with linear DNA:
ligase  introduction of Infection in paralel with production of a catenane production of a catenane
the fragment in the M13 phage to allow phage and in vitro packaging and in vitro packaging
polylinker assembly
End result Colonies Plaques Plaques Plaques Colonies
Uses Obtaining ssDNA (site- Obtaining ssDNA (site- Genomic libraries Genomic libraries
directed mutagenesis and directed mutagenesis and
sequencing) sequencing)

20
For yeast

The discovery of the 2 µm plasmid (6 kb) enabled the production of many types of yeast
vectors. This vector is present in S. cerevisiae in 70-200 copies/cell; it contains a yeast ori.
The selection markers used in yeast vectors are auxotrophic: genes involved in the synthesis of
a compound that is essential for yeast growth. An example is the LEU gene, carried in the
vectors. After transformation the cells are plated in minimal medium without leucine and only
the transformants grow. In order to this selection marker to work, leu2- strains must be used
(auxotrophic host).

Yeast episomal plasmids (YEps)

These are shuttle vectors: they can replicate in E.coli
and S.cerevisiae and have 20-50 copies/cell.
These vectors are hybrids, containing:
 Whole pBR322 sequence (containing 2
resistance genes)
o ampR
o tetR: where the restriction
sequences are
 Where the fragment will insert
 Has the bacterial ori, allowing for replication in bacterial cells
 Whole or partial 2 µm plasmid sequence (containing the ori for yeast)
 LEU2 gene from yeast chromosomal DNA

Prior to transformation in yeast, the vectors can be

inserted in E.coli to select the recombinant clones and
purify the recombinant plasmids. This approach is
useful since there’s more information available on
E.coli and it’s easier to extract the plasmids from them.

Episome = ability to replicate or integrate

A YEp can integrate the genome by chromosomal
recombination. With YEp13, recombination is done
between the LEU2 in the plasmid and the mutated non
functional LEU2 in the yeast genome.

21
Yeast integrative plasmids (YIps)
They’re bacterial plasmid with an inserted yeast gene, allowing for homologous recombination
with the yeast genome.They only survive by integrating, since they don’t have a yeast ori and
can’t replicate.

Since yeast have lots of transposons, when a YIp is inserted the gene
of interest might replicate in the genome.
To increase the number of copies of the target gene, this might be
inserted in tandem in the plasmid.
The selection is the same as with YEps.

Yeast replicative plasmids (YRps)

Plasmids that carry a yeast ori, having 5-100 copies/cell.
They’re unstable because they might get lost  During budding the
plasmid might be left in the mother cell. Then we have 1 cell with the
plasmid and one without it. The one with it will divide more slowly
due to extra energy expenditure involved in the plasmid replication.
This gives an advantage to the yeast cell without the plasmids and
might result in loss of the plasmid in the population.
However, these plasmids are still used to produce proteins because
they have high copies numbers per cell.

Factors important for choosing the plasmid:

 Transformation frequency: YEps> YRps > YIps (this is because integration is needed for
plasmid survival and this is a rare event)
 Copy number: YEps (20-50), YRps (5-100) > YIps (1)
o Important if we want to produce a protein
 Stability: YIps > YEps > YRps (loss of the plasmid during budding)

Yeast artificial chromosomes (YACs)

Multiple yeast genes were cloned into a plasmid.
 Centromere (CEN4)
 Yeast ori
 Telomeres (TEL)
 Selection markers
o Auxotrophic: TRP1, URA3
o For the selection of recombinants:
SUP4 (produces a blue pigment)

22
The plasmid is treated with BamHI and SnaBI. The BamHI opens the arms, leaving the TEL
sequences at the extremities. The SnaBI opens in the SUP4 gene, leaving blunt ends for
inserting the fragment of interest.
The strains must be trp1- ura3-.
The selection is done in minimal medium:
 Transformants with ligations between
both left or both right arms  won’t
grow because they only have 1 of the
auxotrophic markers.
 The right recombinants are white,
don’t produce the pigments

These vectors can be used to clone large

fragments, which is important in the study of
mammalian genes. They are also used in gene
libraries: up to 1400 Kb. However, the YACs that can harbor these larger fragments have
stability problems, due to intramolecular recombination.

For plants
TI plasmids
This is a conjugative transfer, similar to bacterial conjugation. The only part that is transferred
is the T-DNA.
This plasmid has a large size, which means that the efficiency of transformation is low and that
there’s no unique restriction site. So, 2 strategies were developed to solve the problem.

Binary vector strategy

This strategy is based on the fact that the T-DNA doesn’t need to be in the same plasmid that
the vir genes.
To solve this, we can use the binary vector strategy  Plasmid A has the vir genes, responsible
for the DNA transfer. The plasmid
B has T-DNA, which has the
fragment of interest, and that is
going to be inserted in the
genome.
So the 2 vectors are introduced in
the plant cells at the same time,
being the plasmid A an helper
plasmid.

23
Cointegration strategy
In this strategy 2 plasmids are inserted in a
plant cell. One is the normal TI plasmid. The
other is small E. coli derived vector that has a
small part of the T-DNA. This allows
recombination between the 2 plasmids,
generating a new recombinant plasmid
within the cell (the recombination happens in
the plant cell!). This recombinant plasmid has
the whole E.coli vector inserted in the T-DNA.
So, all of this will be inserted in the plant
genome.

Disarmed TI plasmids
TI plasmids are used to transform plant protoplast and
then grow a transformed plant. These plasmids are
“disarmed”: the T-DNA doesn’t have the genes involved
in tumor formation.

Since the only parts in T-DNA necessary for infection

are the 25 bp flanking repeats, a new binary vector can
be made. It has a selectable marker, like kan, and a lacZ
where the gene will be inserted.

RI plasmid
It’s similar to the TI-plasmid, except that it causes hairy root disease instead of crown gall
disease. This can be used to produce proteins in the roots in liquid culture (the target gene
might be put in a promoter that is active in the roots). However, there’s only 1 copy/cell.

The use of RI and TI plasmids is exclusive to dicotyledonous plants.

Direct gene transfer

Supercoiled plasmids are used. These can’t replicate but the DNA integrates the host genome.
The recombination process isn’t known.

24
Gene transfer into the chloroplast genome

The vector has the LTR and RTR sequences, which are also present in the chloroplast genome.
These sequences flank the region which contains the gene of interest and selectable marker,
like an antibiotic. This region is inserted by homologous recombination.
Since a plant cell has an average of 10 chloroplasts, this is a good strategy to increase
expression.
The transformation is done in embryos that are treated with the antibiotic for a while to allow
the transformed genome to propagate.

For insects
A normal P element has terminal inverted repeats that allow for the fragment to jump and
genes encoding transposase.

The plasmid contains 2 P elements:

 R  P-element which has the
terminal inverted repeats (can
jump) but that has an inactivated
transposase gene. This is where
the target gene is inserted.
 “Wings-clipped” element 
doesn’t have the terminal inverted
repeats (can’t jump) but has the
transposase gene.
This is done in a way that when the R
fragment is inserted in the fly genome it
becomes stable, since it doesn’t encode
for transposase.

25
Polymerase Chain Reaction

Primers

Degenerated primers
They’re used when the gene to be amplified is unknown but we have at least homologs protein
sequence. The aa sequence is used because it is conserved and the nucleotide sequence isn’t.

(a) Doing a multiple

alignment we can identify
the conserved regions.
Within these regions we
choose one that has few aas
that have lots of codon
choices (ex: leucine)
(b) Find the nº of
possibilities for that regions
(c) Use inosine for the
rd
3 base in the codons that
have 4 possibilities. That
way the nº of possibilities
decreases and we have to
use fewer primers.

Using this approach we

can’t amplify the whole
gene, only the conserved
region. To find out the gene,
we do a southern blot of the amplified fragment against gene bank samples.

26
Rules to design primers
 Length: primers should have 17-28 bps
o If they’re too short they might have lots of hybridization sites due to chance
4(primer length)/size of the genome = nº of possible sites to hibridize
o If they’re too long they take too much time to hybridize
 Melting temperature between 55-80ºC
o Annealing temperature  If it’s too high the primers won’t hybridize and if it’s
too low they will hybridize with low specificity
Tanneal=Tmelting – 5ºC
o Tm, melting temperature
Tm= (4x(G+C)) + (2x (A+T)) ºC
Tm is the temperature at which half of the primers are ss and half are ds.
We can’t use primers with big differences in the Tm because will have very different annealing
efficiencies. Ex: If Tm(primer1)= 55ºC; Tm(primer2)=66ºC and we use annealing temp= 56ºC 
The primer1 will hybridize much more efficiently than the primer2. If we use 65ºC the primer1
may not hybridize at all.
So, we have to choose the right primer size to adapt the Tm
 GC content ~50-60%  If it’s higher we may have too much thermal stability (higher
Tm)
 3’ end should be G/C/GC/CG: increases the priming efficiency because the polymerase
binds better (?)
 Primers shouldn’t be complementary  They’ll hybridize and will be amplified much
more efficiently than the target fragment
 They mustn’t have too much C/Gs at the 3’ end  The primers may hybridize with
regions that are only complementary at the 3’ end and create unspecific amplicons

Taq Polymerase
Doesn’t have a proofreading activity : error rate of 1/9000  1 error/300 bps after 30 cycles
Adds an A to the 3’ ends  We can use exonucleases to take it out or vectors that have a T-tail

Using PCR products for cloning

 Primers that contain restriction sequences  After amplification we can use that
restriction endonuclease to create sticky ends in the primer sequence

Applications:

-Cloning of genes (gene discovery)

-Sequence determination

-Site-directed mutagenesis

-Gene expression (RT-PCR, real time-PCR)

-Molecular typing (ribotyping, RAPD)

27
-Taxonomy

-Diagnostic of pathogens

-Forensic medicine (genotyping)

How to obtain a clone of a specific

gene
After cloning we have to select just the colonies
that have the vector with the gene of interest
inserted. This is because after transformation we
have cultures that have the vector with other
fragments inserted.
There are two main strategies to do this:
 Direct selection  This is when the cloning
was designed in a way that the
recombinants will have a phenotype that
can be identified in the media (ex:
antibiotic resistance).
 Identification of the clone from a gene
library  This is when we do an initial
cloning where we introduce all or most of the genes, creating a library.

In the 1st strategy selection markers can be antibiotic resistance or auxotrophic (marker
rescue). One example of these 2nd type of markers is trpA, the gene encoding tryptophan
synthase. We have to use parental strains that are trpA- and plate the transformants in
minimal medium without tryptophan. This means that the auxotroph strain and the minimal
medium must be available.

Identification of a clone from a

gene library
Genome libraries are created for
prokaryotes and yeast.
1. Digest the genome with a
restriction endonuclease,
generating 35 Kb random
fragments.
2. Ligate the fragments in cosmids.
3. Transform

28
Colonies with 35 Kb fragments representing the whole genome are obtained.

In other eukaryotes gene libraries are created. These are specific for different cell types, since
different tissues have different expression patterns.
1. Extract mRNA
2. Use poly-T primers that anneal with the poly-A tail of
mRNAs
3. Use reverse transcriptase to synthesize the 1st strand
of DNA
4. Use RNAses to degrade the mRNA, leaving behind
some RNA fragments attached to the DNA strand,
that will act as primers
5. Synthesize the 2nd DNA strand
6. Using TdT and only one dNTP create a homopolymer
tail. Do the same for the vector. Ligate.
7. Transform the bacteria and plate.
This way we get colonies that represent all the mRNA in the
cell. The nº of colonies with a given cDNA will depend on the
nº of mRNA molecules in the cell.

A desired clone is identified by hybridization probing

(southern hybridization) or PCR.

Southern hybridization
Principle  In this technique the target DNA is immobilized
and the probe is added. If there’s extensive complementarity
the probe will be bound to the sample. Then a colorless
substrate is added and the enzyme bound to the probe will
catalyze its conversion to a colored precipitate. Radioactive
probes can also be used and in that case.

In this approach we need a probe so we have to at least find a

homolog in another species (a hybrid doesn’t have to be 100% complementary to be stable).

Procedure:
1. Treat the genomic DNA with a restriction endonuclease. Separate the fragments by
electrophoresis.
2. Treat the gel with acid to depurinate
(nicks in the DNA) and sodium
hydroxide to denature .
3. Transfer the fragments to a
nitrocellulose membrane: it has a
positive charge so the phosphate

29
 groups of the DNA will bind. There are 2 systems used to do this:
 Capillary transfer  The weight on top exerts pressure on the system. A buffer
gradient is used so that it moves from down to up, taking the DNA with it. The
pores in the membrane don’t allow the DNA to pass through it.
 Vacuum system  The vacuum is on top. It’s a faster process.
4. Expose to UV light to stabilize the DNA and membrane, by making a covalent bond.
5. Use a pre-hybridization buffer with heterologous DNA (salmon sperm) to block the
membrane. Otherwise the probe would bind non-specifically to the positive charge of
the membrane.
6. Hybridize the membrane with the denatured labelled probe. Wash.
7. Add the detection reagents and visualize.

Colony hybridization
This technique is used when we want to identify the clones with a given gene in a gene bank.
The membrane with the bound bacteria is treated like a DNA extract.
Procedure:
1. Transfer the colonies to a nitrocellulose membrane.
2. The membrane is treated with alkali and proteases to degrade all the cellular material,
just leaving the DNA.
3. Use UV light or heat to stabilize the DNA-membrane bond.
4. Add the probe and let it hybridize. Wash.
5. Detection with autoradiography, colorimetry or chemo luminescence.
This strategy gives a lot of false negatives because of all the dirt.

Another possibility is to do the DNA extraction in batches. If we get a positive signal in one of
the batches we divide it in 5 batches. If we get a positive signal in one of them, etc…

Probes
 They have to share at least a part of the sequence of the gene of interest. At least 50%
of homology with the gene we want to identify (ex: we can use algC to find pgmG,
using lower temperatures)
 Size: 100-1000 bps  Too small may hybridize
non-specifically; too big takes too much time and
is inefficient.
 Can be labelled with radioactive or non-
radioactive dNTPs. Nowadays we use non-
radioactive probes: biotin, digoxigenin, fluorescein
The PCR product is denatured with heating and the
temperature (60ºC) is maintained to avoid renaturation.
Random hexamer nucleotides are used as primers, the mix
being complex enough to include at least a few molecules
that bind to the ssDNA and allow the synthesis of the
complementary strand. The Klenow fragment is used to
polymerize. One of the dNTPs used is labelled.

30
The objective of using random primers, instead of specific ones, is that they can be sealed in
commercial kits to use in every sequence.
 If the label is biotin we use avidin coupled with a fluorescent marker to detect

Membranes can be recycled

Wash with buffer (containing SDS) to remove the probe. Then we can start again at the pre-
hybridization step. This is useful when the results don’t make sense and we want to repeat the
experiment without throwing everything away. This can be done ~4 times.

Applications
 Confirm deletion mutants
We use a probe to the surrounding genes and compare the size of the fragments. The mutants
will have smaller fragments due to the absence of the gene. This is used in cases where PCR
doesn’t work: big genes.
 Gene identification: case of pgmG from S.elodea
They used degenerated primers based on proteins with phosphoglucomutase activity to
amplify the internal fragments of pgmG and use it as probe. They then did a screening of the
S.elodea genomic library.

Mutagenesis and interference

In bacteria
Transposon mutagenesis
(random)

Transposons can move around in the genome or copy and then jum.
Plasposons are used: in addition to the transposase gene and the selectable marker, they
contain the bacterial ori within the inverted repeats.

In this strategy, transposons are used to randomly mutate genes. Then all the colonies are
analyzed for some phenotype (forward genetics)

Example: if we want to find the genes in B.cepacia involved in exopolysaccharide production.

31
The plasposon was transferred to B.cepacia by triparental conjugation (it was mob+). The
transformants were selected using a medium supplemented with kanamycin. The nonmucoid
colonies were selected using sudan black staining.
So this way we get all the mutants that can’t produce exopolysaccharide. How can we know
where the transposon inserted?
DNA is extracted and restricted.
The fragments and ligated into
vectors and they’re used to
clone E.coli, which is grown in
medium supplemented with
kanamycin. Only the clones with
the region from the transposon
will have the kan gene and be
able to grown in that medium.
These colonies are selected and
the plasmids are sequenced.

What if we want to discard strains that have 2 transposons inserted?

Do a Southern blot, the probe is the plasposon. If there’re 2 transposons inserted there will be
2 fragments with different sizes.

This method has a lot of steps, which means it has low efficiency.
It can also have errors associated. For example, if a transposon inserts in the middle of an
operon it will affect all the genes from then on. So we can’t say the phenotype is only due to
the interference in the gene it inserted.

Deletion of genes
It uses a recombination strategy.

There are 2 recombination events: in

the 1st the shuttle vector integrates
the genome due to homology in the A
region. In the 2nd the vectors is excised
due to homology in the B region.

32
It has to be a suicide vector: unable to replicate in the organism we want to mutate, although
it can replicate in E.coli.
It has 2 selection markers: kan and gusA (produces a pigment). This cassette is flanked by
regions similar to the ones flanking the gene we want to mutate.

Strategy of 2 recombination rounds

So the single recombinants are

WTs that have the Km gene.
The double recombinants are WT
or deletion mutants.

Since single recombinants are

much more likely to happen than
double recombinants we use 2
rounds of recombination. Also, we
can’t distinguish WT from deletion
mutants among the double
recombinants.

The rounds of recombination are

induced by exposing the cells to
stress.

1st round  There are 3 possible outcomes:

 Single recombinations: A-A and C-C  Resistant to kanamycin
 Double recombinants  Susceptible to kanamycin
 Non recombinants  Susceptible to kanamycin
So we plate in medium supplemented with kanamycin to select only the single recombinants.
Then we induce the 2nd round of recombinantion.

2nd round of recombination  3 possible outcomes:

 Didn’t suffer a 2nd recombination event: single recombinants  They have gusA so
produce the pigment
 Suffered a 2nd recombination event: double recombinants  White colonies
o WT: suffered 2 rounds of A-A recombination or 2 round of C-C recombination

33
 o Deletion mutants: suffered a 1st A-A recombination and a 2nd C-C
recombination (or vice-versa)

To distinguish the deletion mutants from the WT, PCR or Southern can be used. In the case of
Southern, the probe has to include AB, so that the WT has a signal and the mutant doesn’t.

 Double recombinants Susceptible

1st round  Non-transformants (WT) to kan
 Single recombinants
Colored
 Single recombinants Resistant  Double recombinants
colonies
2nd round
to kan o WT White
o Mutant colonies

Distinguish with PCR or Southern

Construction of the plasmids

We want to get a vector with an AC fragments (flanking regions) and 2 selectable markers.
We have vectors with 2 selectable markers (for example: gusA and neo) and a lacZ gene with a
multicloning site.

1st strategy
Do a PCR to isolate both the A and the C fragment. The primers have to be designed so that we
get the right recognition sites flanking the fragments:

X recognition Y recognition Y recognition Z recognition

A fragment site
C fragment site
site site

Open the vector at the multicloning site with X and Z enzymes. Do a triple ligation of the A
fragment, C fragment and vector and select the white colonies in medium supplemented with
neomycin and X-GAL (the lacZ was disrupted).
 The triple ligation step is very inefficient: the right ligation is very unlikely to happen.

2nd strategy
Also do a PCR to isolate the A and C fragments as above.
But the ligation step is in 2 rounds. In the 1st introduce the A fragment and select the white
colonies in LB medium with neomycin and gusA. In the 2nd round introduce the C fragment. I n
this round there’s no selection marker, because lacZ was already disrupted. So to distinguish
the clones with A-C fragments from those with just the A fragment we have to use PCR or
southern.

34
Marked mutants
The deletion mutants above are unmarked. With this strategy we insert an interposon that
contains a resistance cassette in the middle of the original gene.

Construction of the plasmid

We can build a normal unmarked vector and then insert the resistance cassette in the middle
of A and C. The selection marker is the antibiotic resistance protein encoded by the cassette.

Making the mutants

Using a vector that contains a gentamycin cassette between the AC fragments and a
tetracycline marker somewhere else. In just one round:
 Single recombinants: resistant to gentamycin and tetracycline
 Double recombinants: only resistant to gentamycin
 Non transformants (WT): susceptible to both
So these mutants take less time to make. However, the presence of the resistance cassette can
cause problems in some biotechnological applications.

Do these cassettes prevent the transcriptions of downstream genes is the same operon?
No, because they carry their own promoters, that induce the transcription of the cassette and
the gene downstream.
(in unmarked deletion mutants the transcription of downstream genes isn’t affected too)

Another approach to marked deletion mutants

35
Instead of growing the cell in stress conditions to induce recombination, DNA damage (cutting)
is used to induce it. The vector contains an I-SceI recognition site, that doesn’t exist in the host
genome.
After the 1st round of recombination the single recombinants are selected. Then a plasmid
encoding the I-SceI endonuclease is introduced and it cuts the DNA. The 2 fragments then
recombine, which can originate WT (if the 2nd recombination is equal to the 1st) or marked
deletion mutants that can be selected through the resistance cassette.

Insertional inactivation

There’s only one recombination

event, which leads to the
insertion of the whole vector in
the gene, disrupting it.

These mutants are only stable

under selective pressure (in this
case in neomycin medium). This
is because without it there’s
always pressure to recover gene function and lose the vector.
However, they’re useful in the initial stages of investigation, when we want establish a relation
between gene and phenotype (reverse genetics), since they’re easy to construct.

In yeast
Single or multiple deletion mutants

The theoretical principle is the same as above: recombination. However, this is more efficient
is yeast and we don’t need long flanking regions (40 bps is
enough).

Deletion with a replacement for a cassette

Use a plasmid that has the kan gene and do a PCR with
primers that will amplify the region containing the cassette.
Add to this primers 40 nucleotides that correspond to the
homology region of the gene we want to target.
The amplicons can be inserted directly in the yeast cells,
there’s no need for cloning.
Select the colonies that have the resistance and confirm by
PCR that the correct gene was deleted.

36
The easiness of this process allows systematic gene targeting, creating deletion mutants
collections (like EUROSCARF).

Double deletion mutants

We can use another cassette. The problem is that there aren’t a lot of cassettes available. So,
we use the Cre-lox technology:

Cre is a recombinase enzyme that acts on lopX sites.

So if we put loxP sites in the regions flanking the cassette, we can take it out anytime we want,
just by adding a plasmid containing the Cre gene. This leaves a loxP site in the region were
there was supposed to be the targeted gene. This way, we can do new mutations in that strain,
using the same cassette.

Testing essential genes

To test if the gene is essential diploid strains are

used.
The cassette replaces one of the copies. Then the
strains undergo sporulation: 50% of the spores will
be WT and 50% will have the cassette instead of
the gene.
So, if the gene is essential only 50% of the offspring
will survive.

37
siRNA interference
Transient mutants

sRNAs bind to mRNAs and induce:

 Post-transcriptional silencing: affecting mRNA stability or preventing translation
 Transcriptional silencing: the siRNA-mRNA is recognized by enzymes that modify the
DNA (histones, methylation, etc), preventing transcription

siRNAs
1. dsRNA is recognized by DICER proteins, that cleaves it into small dsRNAs
2. these associate with ARGO (RISK in animals), creating ssRNA-ARGO complexes
3. these complexes have silencing activity
These act on transcriptional and post-transcriptional silencing and are involved in stress and
defense responses.

How does dsRNA appear in the cell?

 Regions where there’s an overlap between genes
dsRNA
coded in opposite strands. The overlapping region
will create dsRNA. (image in the left)
 Viral infection
o Most plant viruses are RNA virus. The replication cycle includes the formation
of dsRNA. So, siRNAs silence virus replication and expression.
 Internal RNA-dependent RNA polymerases that synthetize dsRNA from ssRNA

microRNAs
There are encoded by the MIR genes.
They have complementary regions,
which allows the bending and then
formation of dsRNA
They bind to mRNAs, slicing them and
inducing translational repression.

Using siRNA only works on higher eukaryotes that have the RISK or ARGO complex.

38
Site-directed mutagenesis
It can be used to insert multiple point mutations, insert new nucleotides or delete them:

The basic technique: single primer method

An ss M13 vector with the
gene of interest is amplified
using a mutated primer. This
creates a ds M13 vector, with
one original strand and one
mutated strand. Then the
vector is inserted in E. coli and
it repairs one of the strands. In
theory, this would mean that
50% of the resulting vectors
would be mutated, since E.
coli would recognize the
mutated strand as the right
one. However, E. coli
recognizes the unmethylated DNA as the wrong one, so it keeps the original strand and repairs
the mutated one. A way to circumvent this problem is to use E. coli strains that are mutant for
these enzymes.
However, this technique still has the problem of needing ssDNA as template and of
contamination: the template DNA contains the unmutated strands and even ds original
strands. This contamination
decreases the proportion of
the progeny that will be
mutated.

The actual technique

A ds vector with the gene is
the template. To amplify,

39
complementary primers are used so that we get one ds mutated vector. After that, DpnI is
added to cleave the template vector, since it recognizes methylated DNA.

The resulting ds DNA has a nick in the region where the primers annealed, so it’s in the linear
form. The addition of a ligase solves this.
The bigger the mutations we want to insert, the more cycles we need to use in the PCR. This is
because the annealing is less efficient.
The primers should have 25-35 bps and the mutation should be in the middle.

Overview
Deletion mutants
Deletion mutants in higher eukaryotes are very difficult to achieve, since there’re high
probabilities that the cassette will insert randomly in the genome, instead of the place it was
supposed to recombine.
The exception is the mouse, where lots of mutant strains have been built, through the use of
mouse embrionary stem cells.

Random mutagenesis
Genome-wide random mutagenesis is applicable to every organism. This can generate tagged
or untagged mutants. Tagged mutants can be used to recover the flanking sequences, using
PCR or hybridization with the inserted sequence. This is useful to generate databases.
Genome-wide random mutagenesis in vertebrates is done through the use of transposons.
In plants, it can be done with transposons or TI-plasmid techniques. There’re maps of
mutations for A. thaliana and M. truncatula and also culture libraries.

sRNA
RNA interference has been used to create libraries of phenocopies. These are phenotypes
generated through the interference in mRNA, while the DNA sequence remains unchaged.
The libraries started in C. elegans but have been expanded to Drosophila, plants, mouse and
human cells.

40
DNA sequencing
Chain termination method
A ssDNA template is needed: a vector with the target
gene inserted or just the gene.
The primers anneal in the region adjacent to the
polylinker (to make sure that we sequence the whole
gene).
There are 4 reactions in parallel. In each reaction
there are four deoxynucleotides (dTTP, dATP, dCTP,
dGTP) and one labeled dideoxynucleotide, that is
different in each parallel reaction. The
dideoxynucleotide doesn’t have an OH group in the
3’ position, which means it blocks the synthesis from
the moment it is incorporated. So, in each reaction,
we get strands with variable lengths that match the
places where the dideoxynucleotide was inserted.
The results of these reactions are separated in a
polyacrylamide gel with urea and high voltage (in
different lanes) and visualized by autoradiography
(the labels are normally radioactive).

Automated DNA sequencing

It was implemented since the 80s.
Instead of 4 parallel reactions, only one is
needed, since 4 different labels are used for
the 4 different dideoxynucleotides.
So, as the gel runs, there’s a static laser that
excites the fluorophores and records the
signal. In the end a chromatogram is
obtained, with the corresponding peaks for
each position.

41
Thermal cycle sequencing Why does the nº of chains
This is a modified version of the original Sanger sequencing method. increase linearly?
The difference lies in the fact that a thermostable polymerase is used, Because only one strand is the
which means we can do many cycles of sequencing. In each cycle the template and this strand isn’t
nº of chains increases in a linear way (not exponential!), which means amplified. Only the
this method can be employed with low amounts of template DNA. complementary strands are
Basically, this method joins the PCR step with sequencing step. synthetized.

Capillary DNA sequencing

In this method, instead of the traditional polyacrylamide gel, glass capillaries filled with the gel
are used. This allows more automation of the process, since we can have many capillaries
“working” at the same time.

Chemical degradation method

It is used when the template DNA forms
intrastrand base pairs and is impossible to
obtain an accurate sequence by the chain
termination method.

42
The DNA is digested with restriction
endonucleases and denatured. A
polynucleotide kinase is used to add a
radioactive nucleotide to the 5´ends of the
strands. The addition of DMSO prevents the
formation of secondary structures. The
strands are separated by electrophoresis: one
is heavier than the other. One of the strands is
purified and will be the sample for sequencing.
The purified strand is used in 4 separate
reactions. In each reaction a different reagent
is added that will only cut in one type of
nucleotide (ex: it only cuts the strands on
positions with adenines). The reaction time is
controlled so that we only have one nick per
strand. The resulting nicked strands are
separated by electrophoresis. Only the
fragments with the 5’ end labelled will be
visualized, which allows the sequencing.

It is difficult to obtain just one nick per strand so this isn’t a very used technique but it’s still
helpful with DNA molecules that create secondary structures after denaturation.

Sequencing genomes

To large fragments DNA molecules we have to

break them in overlapping fragments and clone the
fragments in vectors, creating a library. Then
sequence each fragment and use an assembly
software.

Applications of massively parallel sequencing

- Mutation discovery
- Sequencing clinical isolates in strain-to-reference
comparisons
- Metagenomics
- Defining DNA–protein interactions
- Exploring chromatin packaging
- Discovering noncoding RNAs

43
Studying gene expression and function
RNA quality has to be checked between all steps to make sure it isn’t degraded. We can do this
with electrophoresis or with an automatized method (Bioanalyzer).

Electrophoresis
If we find the bands correspondent to rRNA (16S and 23S in bacteria, 18S and 28S in
eukaryotes) it means the RNA isn’t degraded.
Disadvantages :
 Time: it takes 24 hours because we can’t have high voltage to don’t overheat the
samples
 Uses formaldehyde and ethidium bromide, which are toxic
 We need to use a part of the sample. If we are doing expression studies our samples
are small.

Bioanalyzer
It works like an electrophoresis but is done microscopically. It has micro channels filled with a
polymer and a fluorescent dye that intercalates the RNA. The sample components are
separated electrophoretically and they pass through a detector, which registers the peaks. It
can analyze 16 samples at once.

Top image: the RNA isn’t

degraded; there are big
and discrete peaks.

Bottom image: the RNA is

degraded because we can
see a lot of small and
overlapping peaks

In this technique small samples can be used (1 µl). This method is also cheap and quick (1
minute).

44
Northern hybridization
This is the RNA equivalent to Southern blot. The RNA extract is separated by electrophoresis
and the bands are transferred from the gel to a nylon/nitrocellulose membrane. A labelled
probe is added and hybridization occurs.
Applications:
 To see if a gene is expressed and how much it is expressed  A housekeeping gene is
used as a control, to make sure that the RNA sample isn’t degraded and to have a
normalization method
 Confirming an operonic structure  There should be a single transcript with the size
corresponding to the sum of all the genes. Any of the genes can be used as probe
(probes have to be < 1kb)

Nuclease protection assay

A labelled RNA probe is hybridized with total RNA. The RNAse only cut ss RNA, which means
that if there’s hybridization the probe won’t de degraded. This allows detection of
complementary sequences in the RNA extract. Since the binding between the probe and target
is stoichiometric, it also allows the quantification of target molecules in the RNA extract (by
using decreasing amounts of RNA extract).

Labels can be radioactive or non-radioactive (ex: biotin).

The bands are transferred to a membrane to allow visualization.
This procedure takes the same time as a northern blot.

Quantitative reverse transcription PCR

Quantitative real-time PCR (RT-PCR)

The 1st step is synthesis of cDNAs for all the transcripts in the extract. If we are studying
eukaryotes poly-T primers can be used; if we’re studying bacteria random primers are used. If
we’re just interest in the expression of one gene we use a specific primer.

The 2nd step is amplification.

45
The fluorescence is measured after each cycle, being proportional to number of copies.
The amplification graph has 3 stages: baseline, exponential and final, where the reaction
efficiency declines.
In the 1st cycles (baseline stage) the signal is too low and below the threshold: the machine
can’t detect those small differences.
The CT value is the cycle number where the fluorescence is higher than the threshold. It
depends on the initial amount of copies. So, if we have a low nº of initial copies, it will take
more cycles for the fluorescence to increase enough and the CT value will be higher.

If we just want to compare gene

expressions the CT value is enough.
If we want to quantify the
expression, we use a graph log
[initial cDNA concentration] vs CT
value.

How do we generate the

fluorescence?
SYBR Green Fluorescence  It
intercalates the DNA and after each
denaturation cycle it is released,
and binds again at the end of the
cycle. Specificity is achieved by
doing separate amplification
reactions for each cDNA.

Taqman probes  These are

specific for each gene. They have an

46
oligonucleotide complementary to the gene, a reporter and a quencher, which blocks the
reporter. When the polymerase (which has 5’-3’ exonuclease activity) gets to the probe, it
degrades the 5’end of it, releasing the reporter, which generatedsfluorescence. This is
measured before the denaturation step.
This technique is more expensive.

Molecular beacons  These are also specific. It

has a secondary structure that enables the
quencher to be close to the reporter, blocking it.
When it hybridizes with the target DNA the
quencher and reporter are far away enough for
the reporter to emit fluorescence. This is
measured in the annealing step.

In multiplex PCR we analyze all the cDNA samples

at once because they have different labels. This
means that we can compare the CT values of each gene, since they were obtained in the same
reaction.

If we amplify each cDNA separately we need to normalize with the CT value of a housekeeping
gene.
( ) ( ) ( )

We need specialized machines for this technique.

47
Reporter genes

We measure the force of the promoter.

Method: Open the plasmid with restriction nucleases upstream of lacZ. Amplify the promoter
using primers that contain the restriction sequences. Then ligate the plasmid and the
promoter, obtaining a plasmid containing the lacZ with the promoter of interest.

Reporter genes have the advantage of not needing specialized equipment (only a
spectrophotometer) and of not involving RNA extraction, which is always a problem.
However, the β-galactosidase mRNA may have a different stability than the mRNA of the gene
of interest. This way, this technique doesn’t take into account sRNAs, secondary structures,
etc…

DNA microarrays
Construction

The spotting of the probes in the chip can be done by a “pin and ring” system or by a
piezoelectric printing, which generates higher density microarrays.

48
cDNA microarrays
They contain Expressed Tagged Sequences (ESTs): cDNAs of transcripts. These are used when
the genome sequence isn’t sequenced. These microarrays are of low density.

Preparation of the target molecules

In this method the two samples hybridize with the probes in the same array, one sample being
the control and the other sample being the test.

The RNA is extracted from both conditions and

cDNA pools are created using 2 different labels
(ex: Cy5-dUTP and Cy3-dUTP). Then the cDNA
pools are joined, denatured (heat or alkaline) and
allowed to co-hybridize with the probes in the
chip. After washing the results are obtained by 1st
exciting with the wavelength for one of the
fluorophores and then for the other.
For each of the fluorophores a monochromatic
image is registered. The 2 spectra are then joined
in a merged image.
The readouts have to be treated:
 Exclude “weird “readouts: regions where there were errors in probe spotting
 Subtract the background fluorescence
 Get a normalized ratio: fluorescence (test) / fluorescence (control)
o 1= no change
o >1 increased expression in the test
o <1 decreased expression in the test

One limitation of these arrays is that we don’t get much confidence in the results because
cross-hybridization of the 2 samples may occur. Also, excluding “weird” readouts may
implicate that we get few probes per gene.
One advantage is that we only need to use 3 arrays (3 replicas), instead of 6.

Oligonucleotide arrays
These arrays have multiple probes per gene: ~11 and are directed to the 3’ end of the gene.
For each of these probes there’s a perfect match (PM) and mismatch (MM).
The PM probe is 100% equal to
genomic reference. The MM probe has
a single nucleotide substitution. The
average of the differences among the
set of probes is the value used to
quantify the expression. This is to
reduce background noise and from
cross-hybridization. It also increases
the accuracy and reproducibility.

49
Each probe has ~25 nucleotides.

To do these arrays we need to have the genome sequenced to create 8-15 unique probes per
gene.

Target preparation

The cDNA pool from each condition is generated using random primers (bacteria) or poly-T
primers (eukaryotes). Then labeled cRNAs are created through in vitro transcription with
biotinylated nucleotides. The cRNAs are fragmented to 35-200 bps so that they’re able to
hybridize with the 25 mer probes.

The readouts are obtained after washing by adding streptavidin and measuring the
fluorescence after excitation.
In this technique we use 6 arrays because it’s 3 replicas for each condition and two conditions.
Microarrays allow a global analysis. The protocol is very standardized so results from different
laboratories can be compared. This is particularly important when there’re few samples (ex:
few patients).

50
Expression of recombinant proteins

Overexpression in bacteria
Features of a vector for expression in E.coli
 Regulatory sequence
 Promoter
 Ribosome Binding Site
(RBS) – Shine Delgarno sequence:
the translational beginning is a
few nucleotides downstream
 ORF: it must be right!
 Terminator:
transcriptional end
 Selectable marker
 Ori

Promoters
Weak vs strong  the strength of the promoter is determined by the affinity for the RNA
polymerase sigma unit
If we want to produce high amounts of the protein a strong promoter is indicated. However,
the protein might be toxic or precipitate when it’s in high amounts (if it has hydrophobic
regions). In that case, a weak promoter might be more useful.

Inducible vs repressible: do we want to control when the expression is induced? If the protein
is toxic in low amounts we might want to let the cultures grow 1st and then express the
protein.
 Lac promoter  IPTG induces it, by binding to lacI, which is a repressor that binds to
the lacO promoter. If there’s no IPTG the lacO promoter should be blocked. However,
there’s basal expression.
 Trp promoter We can use 3-β-indoleacrylic acid to induce or tryptophan to repress it
 Tac promoter  Hybrid between lac and trp. It’s as strong as the trp promoter but we
can use IPTG to induce it
 λPL promoter  it is induced by low temperatures. It’s used in industry because it’s
cheaper than using IPTG.
 T7 (from the T7 phage)  For this we have to insert 2 vectors: one with the T7 RNA
polymerase under the control of lacO and other with the gene of interest under the
control of T7. This is because the bacterial RNA polymerase doesn’t bind the T7
promoter and so we have to introduce it the cell. We use IPTG to induce T7 RNA
polymerase expression. In the absence of IPTG there’s no basal expression (some T7
RNA polymerase is produced but it’s not enough to induce the T7 promoter)

51
 Fusion proteins
In this case we use the native elements: promoter, RBS, ATG, a part of the native gene, our
foreign gene and the terminator.

Advantages of the fusion system:

 The start of the coding sequence of the
native gene might improve translation. This is
because the 1st codons of the native gene are
optimized for translation and transcription in
bacteria and our foreign gene isn’t.
 It may also create secondary
structures that cover the RBS!
 The initial bacterial segment might
stabilize the protein and prevent degradation.
 The initial bacterial segment may
include a signal peptide for export, which is
desirable, since it simplifies the problem of
purification.
 However, this creates another
problem: dilution of the protein in the
extracellular medium
 It might help purification by enabling the fusion protein to be recovered by affinity
chromatography.
 In the case of using a tag

Tags
Poly-histidine  A HIS tag located before the multi
cloning site. This way we can recover the protein of
interest with an affinity chromatography with nickel.
What if we don’t get any protein with chromatography?
 The protein fold is hiding the HIS-tag
 The HIS-tag was cleaved with the signal peptide
 It has precipitated: it’s in inclusion bodies

Glutathione-S-Transferase
Example of the PGex-3 plasmid:
GST PreScission protéase seq
The PreScission protease seq is a
MCS
signal sequence for a protease.

Using this fusion protein we can recover it by doing a 1st affinity chromatography with
glutathione (that binds to GST). Then we incubate the fusion protein with the protease,

52
allowing it to cleave the protein. A 2n affinity chromatography with glutathione is done to
“trap” the GST and elute the protein of interest.
This approach is more expensive, so it’s only used when the HIS-tag doesn’t work.

Problems with expression in bacteria

Introns
If the foreign gene has introns; bacteria don’t have the splicing machinery.
 Use cDNA: from a library or extract mRNA to produce it

Translation
The foreign gene might have terminator sequences that form loop structures, stopping
transcription. In this case we obtain a truncated protein. This is a rare phenomenon.
Codon bias: the organism might have few tRNAs for the codons in the foreign gene. In that
case the translation rate is slow.
 Both problems can be solved by site-directed mutagenesis, as long as the aminoacid
sequence isn’t changed.

Post-translational modifications
Dissulfide bonds: bacteria have this machinery in the periplasm. If the recombinant protein is
in the cytoplasm it will be incorrectly folded.
Glycosylation: bacteria don’t have these enzymes.
The wrong folding can cause precipitation in inclusion bodies.
 This can be solved by overexpression of the enzymes: chaperones or
glycosyltransferases
 Inclusion bodies can be recovered with urea. The protein will be in the denatured form
but refolding can be induced with buffers, pH, cofactors, etc… It also depends on the
application: if we want the protein to produce antibodies against it, it can be
denatured.

Degradation: bacteria might have proteases that degrade the protein when it enters stationary
phase or upon environmental changes.
 Use strains that are deficient for the proteases involved

53
Expression in eukaryotes
The 1st options after E. coli is eliminated are yeast and
fungi, because they’re easy to grow. Since they’re
eukaryotes, they have a higher probability of processing
the protein correctly.

Features of a eukaryotic expression vector:

 oriE and Amp  To be able to clone and select the
gene of interest in E.coli (shuttle vector)
 Multiple cloning site with:
o Promoter: normally GAL (induced with
galactose)
o Terminator: animal termination signal doesn’t work well on yeast
 Eukaryotic selectable marker (ESM) with promoter and terminator  It can be
auxotrophic or an antibiotic resistance protein
 ori (euk)  origin of replication for eukaryotes

Expression in Saccharomyces cerevisiae

It’s the most popular microbial eukaryote for production of recombinant proteins. The
medium is similar to LB but richer, which enables the population to grow up to OD=14.
Some problems:
o They hyperglycosylate proteins that have glycosylation signals  The resulting
proteins are immunogenic
o Lack of a secreting system into the growth medium
o Codon bias

Expression in Pichia pastoris

It can grow up to OD=32. The promoter of choice is the AOX, from the alcohol oxidase gene,
which is induced by methanol.
It can glycosylate in similar patterns to animals. However, we might need the glycosylation to
be just right. So, strains were engineered to produce the glycosylation enzymes.
It has a problem associated: some recombinant proteins are degraded.

Expression in mammals
Features of the vector:
It also has the features of a shuttle
vector (oriE+Amp), the MCS with the
flanking regions and a selectable
marker. This can be:
 Puromycing N-
acetyltransferase (pac)  Puromycin
is put in the medium and the

54
 transformants acetylate it, repressing its inhibition in protein synthesis
 Dihydrofolate reductase (djfr)  The transformants metabolize the dihydrofolate,
repressing its inhibition in DNA synthesis

What if the protein is a dimer?

We use an expression system with 2

vectors for the 2 genes. The vectors are
similar, except with the selectable markers,
which are different to allow the selection of
transformants with the 2 vectors.

Some examples

Insulin

Proinsulin is physiologically produced in the inactive

form. Upon cleavage of the C chain, it becomes
activated.

The 1st attempt to produce insulin was in the 70s:

there was no PCR so the gene sequence wasn’t
known, only the aminoacid sequence.
They produced the 2 chains separately in two
cultures and then mixed them. They used the initial
segment of the β-galactosidase gene. However, the
process had a low yield because the disulfide
bridges formed with a low frequency.

Then they tried to clone an artificial gene with the

BCA chain. It folded spontaneously.

55
Growth hormones
The 1st to be produced was somatostatin, in the 70s. They were able to synthesize the artificial
gene, since it was small (14 aminoacids). They used the same strategy as with insulin and it
worked easily because the peptide doesn’t have any post-translational modifications.
Somatrophin was more difficult because it has 191 aminoacids, they couldn’t synthesize the
DNA. So they extracted mRNA from the pituitary and produced a cDNA to clone. It worked
because it also doesn’t have post-translational modifications.

Recombinant factor VIII

The gene has 186 KB and 26 exons. It is
too big to be amplified by PCR and to be
translated in E. coli. It also has post-
translational events.

The 1st attempt was to clone the entire

cDNA in hamster cells but it had low
yield.

They then tried cloning a smaller cDNA:

just the A and C chain. A strong promoter
in hamster cells was used. They could
obtain a high yield and the protein was in
the native form.

Western blot
It can be used to determine the size of the protein, the amount of protein present (semi-
quantitative) and in what tissue it is expressed.

The proteins are treated with SDS and mercaptoethanol, to denature and confer a net negative
charge. They are then run in a SDS-PAGE, separated by size only. The bands are transferred to
a nitrocellulose membrane with an electric field.

The visualization is with antibodies:

 Primary antibody: binds to the protein
o Polyclonal  Different Abs that target different epitopes in the same protein,
they’re cheaper
o Monoclonal  Only one Ab that targets one epitope

56
  Secondary antibody: against Abs produced by the animal from where the primary
antibody was produced
Before addition of the Abs the membrane has to be blocked to prevent non-specific binding of
the Abs.

Proteomics
Proteins expressed in a biological sample at a given time point in a given situation. Proteomics
is thus the study of proteomes.

Three Kinds of Proteomics:

 Expression or Analytical Proteomics - is the quantitative study of protein expression
between samples that differ by some variable
o 2 dimensional electrophoresis gels
o Mass Spectrometry
 Functional or Interaction Proteomics- use of a protein ligand to isolate specific types
of proteins. Is important for signaling or protein-drug interactions
o Protein Domains & motifs
o Post-translational modifications
 Structural proteomics- when the goal is to map out the structure of protein complexes
or the proteins present in a specific cellular organelle (cell map). Determines all
protein-proteininteractions
o High throughput X-ray Crystallography/Modeling
o High throughput NMR Spectroscopy/Modeling

57
Interactomics

Genetic approaches: Two-hybrid systems

Yeast two-hybrid systems

There are two separate vectors that express the fusion proteins. These vectors are selected in
E. coli.
The promoter (Upstream Activated Sequence – UAS) is associated to a reporter gene in the
host genome.

Based on the intensity of the color, which correlates to the β-galactosidase expression level,
we might infer about the strength of the interaction.

To control the experiment we should trade the domains:1st X is bound to PREY and then X is
bound to BAIT. This is to make sure that the interaction doesn’t depend on the domain that
the proteins are bound to.

This system doesn’t work on membrane proteins because they can’t translocate to the
nucleus

Bacteria adenylate cyclase two-hybrid system (BATCH)

It can be used for
membrane proteins
Each protein is
fused to 2
fragments of the
catalytic domain of
adenylate cyclase.

58
When the proteins interact the 2 fragments are Intracellular [cAMP] increases in response to low
brought together and catalyze the conversion of glucose levels. So, cAMP is a secondary messenger
ATP in cAMP, which activates catabolic operons that binds to Catabolite Activator Protein (CAP),
like lacZ. activating the lac operon, among others.

Limitations of two-hybrid systems

It can give a lot of false negatives:
 If the proteins have post-translational modification they might not exist in yeast and
bacteria. This can affect the interaction and give a false negative.
 The interaction might not be strong enough to induce reporter gene expression. It also
might not induce the joining of the 2 domains.
However, these methods give few false positives, since they’re in vivo. The few false positives
can be due to the protein interacting directly with the promoter of the reporter gene (in the
case of yeast two-hybrid systems)

Biochemical approaches
In vitro

Phage display
It allows studying all the interactions with a given protein.
In this method the gene of interest is fused with a phage coat protein gene. This way, the
protein of interest will be displayed on the phage M13. By systematically doing this we
generate a phage display library.

We add a phage display library

to the immobilized protein of
interest. After washing, the
retained phages are recovered
to extract DNA, clone (to
increase the DNA amount) and
then sequence.

Limitations
 We need a phage display library
 It might create false positives: interactions that don’t happen in vivo (ex: proteins that
are in different cell compartments)

One application of this technique is the pharmaceutical area. If we have a drug target we are
trying to find inhibitors: use samples from deep seas to amplify and clone into a phage library.
Then add to the protein of interest to test for interactions.

59
Pull down experiments: co-immunoprecipitation
This is also to test every possible interaction with one specific protein.

A cell lysate is incubated

with agarose beads
coupled to an Ab against
the target protein. After
elution, the proteins
interacting with the
complex Ab-Ag can be
recovered and identified by
MS.

This is a method used in an initial screen. After that, we still test specific interactions with two-
hybrid systems, to confirm that the interaction happens in vivo.
It also has the limitation that we need an Ab specific for this protein only.

Surface Plasmon Resonance (SPR)

We have to know the 2 interaction partners. It doesn’t have to be just protein-protein, we can
test anything that we’re able to purify.
One of the partners is
immobilized in the sensor chip
and the other is soluble in a
buffer. If there’s an interaction
the refractive index changes and
is recorded.
Association and dissociation
values are calculated to
characterize the interaction.
After the procedure, a
regeneration solution is injected
to remove the remaining analyte.
This allows the reutilization of
the immobilized chip: we can test multiple soluble partners for one immobilized one. Each run
takes about 10 minutes.

The kit has a matrix to cross-link one

of the partners. This can be done in 3
ways:
 Directly with the dextran
matrix

60
 Indirectly to a capturing molecule
 Indirectly to a lipid bilayer (for membrane proteins)

Applications:
 Antibody production  In hybridomas different Abs are produced. We can use this to
test the affinity and select the best one.
 Promoters, lipids, etc…
 Finding possible drugs: put a complex mixture and see if any of it interacts with the
drug target
 This is a different and more expensive machine that can fractionate the
mixture and then record which fractions interacted with the immobilized
partner.

The disadvantage of this technique is that we must be able to purify the compounds. With
membrane proteins this can be a challenge.

In vivo: using tags

The protein of interest is cloned with a tag (ex: poly-histidine), overexpressed and put in an
affinity chromatography column for the tag. After washing, the eluted will contain not only the
protein of interest but also the interacting proteins.
Then this fraction is separated and identified by MS.

Tandem Affinity Purification (TAP)

CBP: calmodulin
binding protein

TEV site:
recognition site for
the protease of
the Tobacco Etch
Virus (TEV)

The protein extracts are subjected to affinity chromatographies.

The 1st one is with IgG anti-protein A. This way, the whole fusion protein and the interacting
partners are immobilized. After addition of ETV protease, the bond is cut and the CBP bound
to the protein and its interacting partners are eluted.

61
This fraction is then is subjected to a 2n chromatography with calmodulin beads. The
complexes bind to the beads and after Ca++ chelation (with EGTA), the complexes are eluted.
After that, we still have to recover the interacting proteins and identify them by MS.
The use of this method decreases the nº of false positives that could be due to unspecific
interactions with the nickel column (in the case of using HIS tags).

Molecular typing methods

Methods to differentiate strains.

Areas of application:
- Clinical Microbiology- to control infectious diseases, identify contamination sources, and
transmission routes
- Ecologic studies involving the release of genetically manipulated microorganisms
- Diversity studies to assay genotypic and phenotypic distribution of microorganisms in
environment
- Validation of industrial processes to determine whether the producing microorganism is the
correct one

A good typing system should:

- Be able to differentiate most of the strains
- Be reproducible over time and different laboratories
- Distinguish environmental isolates and not only collections of lab strains
- Be easy to perform and at reduced cost
- Be fast

Genome analysis by pulsed-field gel electrophoresis (PFGE)

The whole genome is digested with an enzyme that makes rare cuts, generating a small
number of big fragments that are then separated by PFGE (Pulsed-Field Gel Electrophoresis).

PFGE
The applied electric field changes 120º from time to time and the fragments need to
reorientate. The bigger ones take more time: this is the retardation time (Tr), which is specific
for fragment size. So, separation is made by retardation.
The critical parameter is the pulse time (Tp), the duration of the electric field in one direction,
before changing. If it is too big (Tp>Tr) it is similar to a conventional electrophoresis. If it is
smaller (Tp<Tr), the fragment moves in the average of the electric fields.
For bigger DNA fragments we use higher switch times.

Preparation of the DNA:

We need intact chromosomes and they’re normally broken by centrifugation, etc…

62
The cells are immobilized in an agarose gel. The lysis buffer is added during 14h (due to
diffusion problems within the agarose). After washing the DNA is digested with restriction
endonucleases. This digestion also takes a long time due to diffusion.

Limitations
 If we have different patterns we can say that they’re different strains but we can’t say
they’re the same strain because they have the same pattern.
 The samples can’t be stored
 This technique takes a long time  Not good for clinical applications
 We need specific equipment
 Small genome changes can’t be detected
 The restriction endonucleases are expensive

Advantages
 The results are reproducible and discriminatory

Ribotyping
This technique is based on the fact that the bacterial genome region that encodes for rRNA has
conserved and variable areas. The conservation is enough to use universal hybridization
probes but the variability allows the different strains to have different restriction sites.

63
So, different strains treated with the same restriction endonuclease will give different
fragments in that genomic area. After hybridization with the universal probes we get different
patterns. Some organisms have more rRNA loci and so the pattern is more complex, which
facilitates the identification.

Technique:
1. Extract total DNA and digest with restriction endonucleases
2. Separate by electrophoresis
3. Denature the DNA and transfer to a nylon membrane
4. Label with the universal probe and detect

This method has the same limitation of the genome analysis by PFGE: we can identify two
different strains but we can’t say that 2 strains are equal. It’s also time consuming.
With the patterns obtained dendograms can be built, which is usefull to understand if different
isolates have a common source.

Advantages:
 It can be used for different organisms
 Commercial probe
 The hybridization patterns are reproducible
 There are databases for comparison

This is the classic way. Nowadays, they just amplify the region, sequence and then blast it.

Randomly amplified polymorphic DNA fingerprints (RAPD)

A PCR is done with a specific set of random primers and the PCR products are separated in a
gel, creating an identifying pattern (RAPD type).

The main advantage is that it is a fast technique: we just have to lyse the cells, extract the
DNA, do the PCR and run the gel (~1 day).
The main disadvantage is that it’s not reproducible, since small differences in the PCR
temperatures will affect the primer annealing efficiency.

Multilocus sequence typing (MLST)

We amplify 7 house-keeping genes that were previously decided for each type of bacteria, so
that they’re present in all strains. The products are then sequenced and the sequence is
compared to databases, where everybody deposits the sequences they’ve collected.
This way, we can not only identify our strain, but also see which strains were already described
and do phylogenetic analysis or population snapshots.

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Epigenetics
Any potentially stable and heritable change in gene expression that occurs without a change in
DNA sequence

Heterochromatin – densely packaged

Euchromatin – less densely packaged

Epigenetics modifications:
 Histone modifications
 Cytosine methylation

Cytosine methylation
(by methyltransferases)

Methylation can be symmetric or asymmetric.

Simmetric methylation is propagated during replication. The 2 daughter DNA molecules only
have 1 strand methylated but the MET1 methylates the other strand (maintenance
methylation).

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Asymmetric methylation requires additional information after replication: one of the daughter
molecules isn’t methylated. This information can be from associated histones or on RNAs
directing methylases to the site.

Methods for studying epigenetic modifications

 DNA methylation: bisulfite sequencing
 Histone modifications
o CHIP
o DNA adenosine methylation identification (DamID)
 siRNA production: deep sequencing (RT-PCR)

DNA methylation
Bissulfide treatment  Extract a sample and treat a part with bisulfite and another don’t. The
treated sample will have the unmethylated Cs replaced by Us (will be read like Ts). The
untreated one will have the unmethylated Cs like Cs. By sequencing the 2 samples we can
know which Cs are methylated and which aren’t.

DNA is more methylated in regions with

repetitive element, like around the
centromere. Mutants for the MET1
methyltransferase have lower DNA
methylation.

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Histone modifications

 Acetylation (on lysines)

 Ubiquitination
 Methylation
 Phosphorylation
 Sumoylation
The position and the type of
modification decide if it’s
transcriptional activation or
inactivation.

Epigenetic controls in whole plant processes

 Transposon silencing
 Control of flowering time
 Developmental switches and stress responses
 Control of imprinted genes
 Gene silencing in trans; paramutation
 Resetting the genome

Transposons
Transposons can copy and move around the genome, being a source of genetic variation. They
can insert into genes, disrupting their function.
60% of the corn genome is transposon DNA. Foreign DNA like this is silenced by epigenetic
modifications like DNA methylation; mutants for these modifications have more mutagenic
transposon activity.

The inactivation of DDM led

to the replication and
insertion of transposons in
new sites throughout the
genome. This caused
abnormal phenotypes.

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How does the genome recognize and silence “non-self” from “self” genes? This is the basis
for genomic immune recognition.
Repetitive elements and transposons are silenced. This requires siRNA activity, which are
preferentially derived from pericentromeric regions.

These siRNAs are produced by DICER and bind ARGO to recruit DNA methylases and histone
modifying enzymes (right).
They can also be produced by RNA-dependent RNA polymerases that synthetize dsRNA that is
posteriorly cut by DICER. The siRNAs then forms a complex with ARGO that binds to RNA and
recruit silencing machinery for the RNA polymerases (left).

Epigenetic modifications in pollination

The sporogenous cells are locate within the anther, which give rise to 4 microspores after the
meiotic division. The callose wall that encloses them is enzymatically degraded and the
microspores are freed. Each of these will form a pollen grain. Through maturation 2 sperm
cells and a vegetative cell will be formed within the pollen grain.

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With pollination, the pollen bursts and the vegetative nucleus dies. A pollen tube is created
and grows to the ovary. There, one sperm cell fuses with egg to form a new plant and the
other sperm cell fuses with the cells to form the placenta.

The main epigenetic modifications are when the sperm cells are in the G1 phase, before
pollination.
By Principal Component analysis (PCA) it was shown that pollen and sperm cells show an
altered transcriptional profile.
DDM1 is more expressed in sperm cells than in pollen.
Transposons are more active in pollen, but in the vegetative nucleus and not in the sperm
cells. So the vegetative nucleus is producing siRNAs that are moved to the sperm cells, where
they inactivate transposons. This results in higher levels of methylation in the sperm cells.

Cystic Fibrosis
Derived from mutations in the CFTR gene, which encodes for a Cl- transporter. It also has
functions in transport of bicarbonate in the lung, GI tract and pancreas and of glutathione in
the lung. The mutations can cause:
 No synthesis
 Defective processing
 Defective regulation
 Altered conductance
 Reduced synthesis
This leads to decreased water content in lung mucus, causing decreased mucociliary clearance
and enabling bacteria to adhere and cause persistent infections, which are accompanied by
inflammation.

Approaches for the investigation of treatment options are of 2 kinds:

 “Bottom-up”: investigate 1st the defect to understand the mechanisms and then
search for therapies that can acts on these mechanisms
 “Top-down”: use drug libraries to find new drugs and targets; just after understand the
mechanisms

The F508del-CFTR mutant

The mutation prevents maturation of the transporter. However, low temperature can rescue
it, enabling its sorting to the membrane.

Proteomics approach
Search for the differences in controls and patients homozygous for the F508del-CFTR
mutation. The results were clustered by metabolic function and 3 main pathways were
identified: chronic inflammation, oxidative stress and cytoskeleton organization.

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Transcriptomics approach
They did the transcriptional profiles of control and mutated with microarrays specific for
human airways tissue. Some of the differentially expressed genes were reanalyzed by RT-PCR.
With this they found the role of the Estrogen Receptor 1 (ER1) in transcriptional control in CF.

Functional Genomics approach

The problem was defined as a reduced membrane content of CFTR and an excess membrane
content of ENaC, which lead to excessive Na+ absorption. So, the goal was to increase CFTR
and decrease ENaC membrane content.

A screening of siRNAs was performed to find possible drug targets. The siRNAs were spotted in
slides and cells were exposed to them. Analysis of CFTR content after exposure to each of
these siRNA enabled the identification of channels inhibitors and activators. This way, genes
involved in CFTR regulation can be identified as potential therapeutic targets.
This was done using strains transformed with the mCherry-Flag-CFTR gene. The mCherry part
is a fluorophore and allows visualization after excitation. The Flag part allows visualization
after addition of anti-Flag Abs bound to
fluorophores.

In the left: WT and mutant in control

conditions. The WT has the transporter both
in the cytoplasm (seen by mCherry) and in
the membrane (seen by mCherring and Abs).
The mutant only has the transporter in the
cytoplasm, which can be seen by the pattern
formed the mCherry fluorescence and by the
absence of signal with the Abs (they can’t
enter the cell).

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Similar strategies were applied to screen siRNAs that affect ENaC activation, using commercial
siRNAs libraries. These were the primary screens, from which 739 genes activating ENaC were
identified. From these, 456 were selected to do a validation screen and 138 genes were
confirmed as activating ENaC.

Molecular imaging
Molecular Imaging emerged in the early twenty-first century as a discipline at the intersection
of molecular biology and in vivo imaging. It enables the visualization of the cellular function
and the follow-up of the molecular process in living organisms without perturbing them.
It can be used in early disease detection.

Medical Imaging techniques

 Computed tomography (CT)  It allows anatomical imaging but can’t be used for
molecular imaging
 Magnetic Resonance Imaging (MRI)  Contrast agents like antibodies or peptides are
used to bind specific targets and alter the image
o Low sensitivity and expensive
o High contrast and spatial resolution
 Optical Imaging (OI)  Using fluorescence, bioluminescence, absorption
o Inexpensive and can be used with a high spatial resolution
o Limited depth penetration

 Ultrasound (US)
o Real time imaging, portable, inexpensive and sensitive
o Whole body imaging not possible, operator dependency, contrast agents only
for vasculature

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  PET and SPECT
o Advantages:
 Unlimited depth penetration
 High sensitivity
 Functional information
 Quantitative molecular imaging
o Disadvantages
 Radiation exposure
 Low spatial resolution
o Multimodality imaging: PET/CT, PET/MRI and SPECT/CT  It allows functional
and anatomical visualization at the same time
The main use is in diagnostic, although there are also applications in therapy.

SPECT and PET probes

Radioactive probes are used. They contain radioactive isotopes, mainly metals.
 1st generation (perfusion agents)  The specificity is dependent on MW, charge and
hydro(lipo)philic nature of the metal complex
 2nd generation (target-specific agents)  The specificity depends on biomolecules to
which the metal complex is attached, like Abs, peptides, etc…

Some examples of perfusion agents:

 Used in bone imaging (bone metastasis)
 Used in brain imaging (strokes)
 Used in nuclear cardiology
o There were some problems of the probes being up taken and metabolized by
the liver, decreasing the cardiac signal  New probes are used, that are
stable, have a high initial and persistent heart uptake, and a rapid blood and
liver clearance (by P-gp efflux). This way, a high heart/liver signal ratio is
achieved

Some examples of molecular imaging probes:

Oncology
Fluorodeoxyglucose(FDG)
It enters the cells and competes with glucose to be
phosphorylated by hexokinase, resulting in the
intracellular accumulation of FDG-6-P, being the
concentration proportional to the rate of glycolysis in the
tissues
 Visualization of tumors with high metabolic
activity : detection and tumor staging, therapy
planning and treatment follow up

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Monoclonal antibodies  They bind to specific Ags that only tumor cells express

Biologically active peptides  Ligands for receptors specific of different tumor types

Cardiology
Atherosclerosis: there’s a need to find diagnostic markers to visualize the plaques and identify
the risk of rupture (the correlation between the degree of obstruction and rupture is low).
This could be done by in vivo identification of the processes underlying progressive plaque
destabilization, like the visualization of the inflammatory activity within the plaque.

Nanobodies
It’s a small fragment of the antibody that maintains the
ability to bind the antigen
.
Conventional Abs have 2 heavy chains and 2 light chains.
The heavy chains have the Vh variable domains and the
light chains have the Vl variable domains, both
necessary for antigen binding and stability.
This technology was developed after the discovery that
the Camelidae can produce Abs that only have 2 heavy
chains. So there’s only one variable domain, the Vhh,
which is stable and maintains the antigen binding
capacity.

How are nanobodies produced?

The camel is immunized to
produce Abs. The mRNAs are
extracted to produce a cDNA
library, which is used to
transform E. coli and later
produce a phage display library.
From that, the phages
displaying the VHH with
specificity to the target Ag are

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selected to clone the DNA and produce the nanobodies.

Nanobodies features:
 High affinity and specificity
 Good solubility and stability
 Small size and fast blood clearance
 Recognition of uncommon or hidden epitopes, binding into cavities or active sites of
enzyme targets
 Efficient radiolabeling

Example: anti-VCAM1 nanobodies  VCAM1 is receptor involved in leukocyte adhesion. These

nanobodies can be used in atherosclerosis imaging.

In vivo imaging of gene expression

Useful for the follow up of gene therapy.

Gene therapy
 Insertion of a normal gene in a specific location to replace a nonfunctional gene
 Replacement of a defective gene by another through homologous recombination
 Repair of the defective gene by selective reverse mutation
 Alteration of the regulation of a particular gene

The most common vectors are altered virus, liposomes or direct insertion of the DNA into the
cells.
 Virus
o Retrovirus  The viral particle is endocytosed and reverse trancriptase
synthesizes ss DNA complementary to the viral genome. From this, dsDNA is
produced and integrates the host genome. Target: dividing cells
o Adenovirus  The viral particle in endocytosed and the viral genome is
imported to the nucleus. The virus uses the cellular machinery to produce RNA
transcripts from its genome. This way, the proteins encoded by the virus are
synthesized. Target: differentiated cells.
 Liposomes  Lipid membranes enclosing a plasmid with gene of interest inserted. The
liposome fuses with the cellular membrane, the plasmid enters the nucleus and the
protein it encodes is synthesized.

The follow up of the treatment can be done with tissue biopsies or assessment on blood
through the evaluation of the expression of marker proteins (co-expressed with the
therapeutic protein).
The tissue biopsy has several disadvantages:
 Risk and patient discomfort
 Change of tissue under study
 Sampling errors due to non-uniform distribution of gene expression in the tissue

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  Limited nº of samples to extract
 The use of histochemical techniques provides limited pharmacokinetic information
 Transfer of non-target cells isn’t controlled
The assessment on blood provides little information about localization and distribution.

So, there’s a need for methods that can evaluate the expression in vivo, providing information
about location, magnitude, and duration. These can involve MRI or PET/SPECT.
 Direct imaging of genes  The probe interacts with the therapeutic target
 Indirect imaging  The probe interacts with the protein encoded by a reporter gene.
The reporter protein can be a:
o Transporter: the probe is a substrate, accumulates intracellularly
o Receptor: the probe is a ligand, accumulates in the cell surface
 Dopamine receptors, peptidic receptors
o Enzyme: the probe is a substrate; metabolic trapping  There’s a signal
amplification because the enzyme can metabolize more than one probe
 Cytosine diaminase, β-galactosidase, tyrosinase, Herpes Simples Virus
1 Timidine Kinase (HSV1-TK)

The HSV1-TK is both a reporter gene and a therapeutic gene. It’s an enzyme that metabolizes a
pro-drug into a toxic compound. So, gene therapy to target cancer cells, combined with
ganciclovir treatment can be used in oncology.

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