18BTC201J Gene Manipulation and

Genomics (Unit 1)

modified on 07/07/2016
INTRODUCTION TO CLONING

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OVERVIEW OF CLONING:

Discovery of enzymes that modify DNA and cut

DNA.
Cutting and joining DNA molecules by restriction
enzymes and ligases, respectively.
Selective amplification of genes of interest using
PCR (Polymerase Chain Reaction) or
Get the DNA fragment with restriction enzyme
digestion.

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Attachment of fragments of DNA to suitable replicons
called cloning vectors.
Small plasmids and bacteriophages are most suitable.
Maintenance does not require integration into the host
genome.
Genetic transformation into Escherichia coli.
Recombinant DNA can be isolated in intact form.

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Pre requisites to gene manipulation:
The vector DNA must be purified and cut open.
The passenger DNA must be inserted into the
vector molecule to create the recombinant DNA.
Cutting and joining reactions are monitored by gel
electrophoresis.
The recombinant must be introduced into the host -
E.coli.

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Detection Techniques:
Agarose and polyacrylamide gels are used to separate
nucleic acids (DNA, RNA).
Northern blot: separation of RNA molecules on
agarose gel, blotting and hybridizing with specific
radioactive or non-radioactive labeled specific probe –
replaced by PCR and real time RT PCR.
Southern blot: separation of DNA molecules on
agarose or polyacrylamide gels, blotting to nylon or
nitrocellulose membranes and hybridizing with
radioactive or non-radioactive labeled specific probe.

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Microarray: a single array or chip may
contain probes to determine transcript
levels for every known gene in the genome
of one or more organisms.
RNA seq (Next Generation Sequencing):
can provide a relative measure of the
cellular concentration of different mRNAs.

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 Gel retardation or band shift assay (EMSA).
Reduction in electrophoretic mobility upon
binding of proteins to DNA.
Co-immunoprecipitation (IP) – to detect protein
and protein interaction.
Chromatin immunoprecipitation (ChIP)-to
detect DNA-protein and protein-protein
interaction in vivo.
Western – Proteins from acrylamide gels to
membranes-detecting specific protein.
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DNA CLONING VECTORS

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Characterization of the DNA of any organism:

DNA cloning: A desired DNA fragment is identified

and selectively amplified so that its structure can be
studied using a variety of different techniques such
as Restriction enzyme analysis, DNA sequencing,
in vitro expression studies.

Molecular Hybridization: The DNA fragment is not

amplified but rather studied as it is found in a
complex mixture of DNA fragments. The restriction
analysis can be performed as well as the
chromosomal location. RNA expression can also be
studied in this way. 10
How to introduce DNA into cells: Vectors

Vectors used to move DNA between species, or

from the lab bench into a living cell, must meet
three requirements:

(1) They must be autonomously replicating

DNA molecules in the host cell.
-The most common vectors are designed for
replicating in bacteria or yeast, but there are
vectors for plants, animals and other species.
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(2) They must contain a selectable marker so
cells containing the recombinant DNA can be
distinguished from those that do not. An
example is drug resistance (tet, amp) in bacteria.

(3) They must have an insertion site to

accommodate foreign DNA.
-Usually a unique restriction cleavage site in a
nonessential region of the vector DNA.
-Recently developed vectors have a set of
about 15 or more unique restriction cleavage
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sites.
How to clone DNA:

1.Cell based DNA cloning.

2. Cell free DNA cloning.

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Cell based Cloning of DNA:
Construction of a recombinant DNA library
Ligation of the target DNA fragments into a suitable
vector.

Transformation of the library into a host

E.coli or yeast to amplify cloned DNA fragments.

Selective propagation of the clones

Plating of clones on selection media to allow
screening.

Isolation of the recombinant clones

Identification and expansion of clones of interest.
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Vectors used in cloning:

1. Plasmids (pBR322, pUC18/19; up to 20kb)

2. Bacteriophages (bacterial viruses; lamda, M13)
(20-50 kb inserts possible)
3. Phagemid (pBS; bluescript)
4. Cosmids (35-50 kb insert)
5. Yeast vectors (2 um; derivatives)
6. Bacterial artificial chromosomes (BACs) (75-300 kb
inserts possible)
7. Yeast artificial chromosomes (YACs) (100-1000 kb
inserts possible).

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 PLASMID CLONING VECTORS

Plasmids are autonomously replicating circular DNA

molecules found in bacteria.
They have their own origin of replication, and they
replicate independently of the origins on the "host"
chromosome.
A plasmid that was widely used in many recombinant
DNA projects is pBR322. It replicates from an origin
derived from a colicin-resistance plasmid (ColE1).

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 This origin allows a fairly high copy number, about 100
copies of the plasmid per cell.
Plasmid pBR322 carries two antibiotic resistance
genes, each derived from different transposons
(jumping genes) that were initially found in R-factors,
which are larger plasmids that confer antibiotic
resistance.

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 Plasmid pBR 322

(beta lactamase)
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Features of plasmid pBR322:
The gene conferring resistance to ampicillin (ApR) can
be interrupted by insertion of a DNA fragment into the
PstI site, and the gene conferring resistance to
tetracycline (TcR) can be interrupted by insertion of a
DNA fragment into the BamHI site.

Use of the TcR and ApR genes allows for easy screening
for recombinants carrying inserts of foreign DNA.

For instance, insertion of a restriction fragment in the

BamHI site of the TcR gene inactivates that gene. One
can still select for ApR colonies, and then screen to see
which ones have lost TcR . 20
Plasmid pUC19 with lacZ gene and Ampr gene and an
E. coli plasmid replicon.

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(IPTG: lacZ inducer
X-gal: substrate for beta galactosidase)
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 Non-recombinant clone
(no cloned DNA insert)

Recombinant clone
(contain cloned DNA insert)

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 Bacteriophages (LAMBDA)
Bacteriophages (lambda and M13) can be used as
cloning vectors.
Lamda phage has a genome of about 50 kb of linear
DNA.
Its life cycle is conducive to the use as a cloning vector.
The lytic cycle can be supported by only a portion of
the genes found in the lambda genome.
This type of lambda vector is also called as the lambda
replacement vector (because the lambda DNA is
removed and replaced with foreign DNA).

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Lambda life cycle:
The lytic life cycle
produces phage
particles
immediately.

The lysogenic life

cycle requires
genes in the middle
of the genome,
which can be
replaced.
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A single lamda genomic DNA is
represented between two cos sites

Endonuclease recognizes the cos sites

and cleaves them.

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Cos sites:
at either end of the lamda DNA molecules
(double stranded), a short 12 nucleotide stretch,
in which DNA is single stranded.

two single strands are complementary and base

pair with one another to form a circular,
completely double stranded molecule.

act as recognition sequences for an endonuclease

cleaves the catenane at the cos sites to produce
individual genomes. 27
LAMBDA INSERTION AND REPLACEMENT
VECTORS

Only 37 to 52 kb DNA fragments can be packaged into

the lambda head.

This can be done in vitro.

The middle portion of the lambda genome can be

replaced (that region is non essential) and DNA can be
cloned (up to 20 kb).

These vectors are used for construction of genomic

DNA libraries.
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 LAMBDA GENOME

Lamda DNA transfection efficiency low; when packed more efficient. 29

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the bacterial chromosome integrated with lamda DNA-lysogen
In vitro packaging systems
Live bacteriophages can be produced by
assembling protein extracts prepared from
bacteriophages with viral DNA
(recombinant) under laboratory conditions.
Live bacteriophages means they are able to
infect bacteria.

Plaques: when bacteriophages

infect and lyse bacteria, a clear
area/zone appears.
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 M13 AND PHAGEMID VECTORS

These vectors are used to produce DNA for sequencing.

M13 is a virus with a genome of single stranded DNA. It

has a nonessential region into which foreign genes can be
inserted.

It has been modified to carry a gene for β‑galactosidase

(lacZ) as a way to screen for recombinants.

The replicative form is duplex, allowing one to cleave

with restriction enzymes and insert foreign DNA.

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 Some vectors are hybrids
between plasmids and
single‑strand phage;
these are called phagemids.

One example is pBluescript.

Phagemids are plasmids (with the modified,

high-copy number ColE1 origin) that also have an
origin of replication of the phage f1 which is related
to phage M 13.
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 The vector has a multiple
cloning site inserted into
the lacZ gene and
which is flanked by
two different phage
RNA polymerase promoters
(T7, SP6).

One can easily obtain either double‑ or

single‑stranded forms of these plasmids.
The "blue" comes from the blue‑white screening for
recombinants that can be done with the multiple
cloning sites which are in the β‑galactosidase gene.
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 The "script" refers to
the ability to make RNA copies
of either strand in vitro
with phage RNA polymerases.

Infection of transformed bacteria (containing the

phagemid) with a helper virus (e.g. derived from
M13) will cause the M13 origin to be activated, and
progeny viruses carrying single‑stranded copies of
the phagemid can be obtained.

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COSMID VECTORS

A cosmid is a hybrid between a lambda vector and

a plasmid.
The COS sites are the only thing that is necessary
for lambda DNA packaging.
Therefore if one can ligate COS sites about 50 kb
apart then the ligation products can be in vitro
packaged.

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 BACTERIOPHAGE P1

These vectors are like lambda and can hold up

to 100 kb of DNA.

This DNA can then be packaged by the P1

phage protein coat.

The use of T4 in vitro packaging systems can

enable the recovery of 100 kb inserts.

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Yeast vectors:

The yeast 2µm plasmid: REP1 and REP2 are involved

in replication of the plasmid, and FLP codes for a
protein that can convert the A form of the plasmid
(shown here) to the B form, in which the gene order has
been rearranged by intramolecular recombination. The
function of D is not exactly known. 40
 Development of cloning vectors for yeast has been
stimulated greatly by the discovery of the 2 µm plasmid
that is present in most strains of S. cerevisiae.

The 2 µm plasmid is an excellent basis for a cloning

vector. It is 6 kb in size which is ideal for a vector, and
exists in the yeast cell at a copy number of between 70
and 200.

Replication: makes use of a plasmid origin, several

enzymes provided by the host cell, and the proteins
coded by the REP1 and REP2 genes carried by the
plasmid.
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 Some yeast cloning vectors carry genes conferring
resistance to inhibitors such as methotrexate and copper
for selection.

Most of the popular yeast vectors make use of a radically

different type of selection system.

In practice a normal yeast gene is used, generally one

that codes for an enzyme involved in amino acid
biosynthesis.

In order to use LEU2 as a selectable marker, a special

kind of host organism is needed. The host must be an
auxotrophic mutant that has a nonfunctional LEU2 gene.
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 Such a leu2- yeast is unable to synthesize leucine and
can survive only if this amino acid is supplied as a
nutrient in the growth medium.

Selection is possible because transformants contain a

plasmid-borne copy of the LEU2 gene, and so are able
to grow in the absence of the amino acid.

In a cloning experiment, cells are plated out onto

minimal medium, which contains no added amino
acids.

Only transformed cells are able to survive and form

colonies. 43
CLONING STRATEGY USING THE YEAST
2 µm PLASMID

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 Yeast 2um plasmid derived:

• Yeast integrating plasmid (YIp):

• Yeast episomal plasmids (YEp)
• Yeast replicating plasmids (YRp)
• Yeast artificial chromosomes (YAC)

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YEPS is a shuttle vector
• Selectable markers, Amp, Tet, Leu2 gene
• Can replicate & can be selected
in both yeast & E. coli
• can integrate with yeast chromosomal DNA

YIPS is a shuttle vector

• Selectable markers, Amp, Tet, URA3 (codes an enzyme
related to the biosynthesis of pyrimidine nucleotides)
• Can replicate & can be selected in both yeast & E. coli
• Is essential to integrate with yeast chromosomal DNA
for survival and replication

YRPS is a shuttle vector

• Selectable markers, Amp, Tet, TRP1
• Can replicate & can be selected
in both yeast & E. coli
• less stable
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YEAST ARTIFICIAL CHROMOSOMES
(YACs)
YACs are yeast vectors with centromeres and
telomeres.

They can carry about 200 kb or larger fragments (in

principle up to 1000 kb = 1 Mb).

Thus very large fragments of DNA can be cloned in

yeast.

In practice, chimeric clones with fragments from

different regions of the genome are obtained fairly
often, and some of the inserts are unstable.
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 Chromosome Structure
Centromere is required for the chromosome to be
distributed correctly to daughter cells during cell
division.
Two telomeres, the structures at the ends of a
chromosome, which are needed in order for the ends
to be replicated correctly and which also prevent the
chromosome from being nibbled away by
exonucleases.
The origins of replication, which are the positions
along the chromosome at which DNA replication
initiates, similar to the origin of replication of a
plasmid.
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Chromosome Structure

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The structure and use of a YAC vector

Several YAC vectors have been

developed but each one
is constructed along
the same lines, with pYAC3 being a typical example.

pYAC3 is essentially a pBR322 plasmid into which a

number of yeast genes have been inserted.

The genes, URA3 from YIp5 and TRP1-ori from

YRp7 have been included in pYAC3.

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 The DNA fragment
containing TRP1 is extended
even further to include the sequence
called CEN4, which is the DNA
from the centromere region
of chromosome 4.
The TRP1–origin–CEN4 fragment therefore contains two
of the three components of the artificial chromosome.
The third component, the telomeres, is provided by the
two sequences called TEL. These are not themselves
complete telomere sequences, but once inside the yeast
nucleus they act as seeding sequences onto which
telomeres will be built.
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 SUP4, is the selectable marker into which new DNA
is inserted during the cloning experiment.

The cloning strategy with pYAC3 is as follows :

The vector is first digested with a combination of

BamHI and SnaBI restriction enzymes, cutting the
molecule into three fragments.

The fragment flanked by

BamHI sites is discarded,
leaving two arms, each bounded by one
TEL sequence and one SnaBI site.
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 The DNA to be cloned, which must have blunt ends
(SnaBI is a blunt end cutter, recognizing the sequence
TACGTA), is ligated between the two arms, producing
the artificial chromosome.

Protoplast transformation is then used to introduce the

artificial chromosome into S. cerevisiae.

The yeast strain that is used is a double auxotrophic

mutant, trp1- ura3-, which is converted to trp1+ ura3+
by the two markers on the artificial
chromosome.

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Any cell transformed with an incorrect artificial
chromosome, containing two left or two right arms
rather than one of each, is not able to grow on
minimal medium as one of the markers is absent.

The presence of the insert DNA in the vector can be

checked by testing for insertional inactivation of
SUP4, which is carried out by a simple colour test:
red colonies are recombinants, white colonies are
not.

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A YAC vector and
the way it is used to
clone large pieces of
DNA.

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