Cloning Vectors

AMIRA A. T. AL-HOSARY
LECTURER OF INFECTIOUS DISEASES
FACULTY OF VET. MEDICINE
ASSIUT UNIVERSITY-EGYPT
 DNA Cloning with Cloning Vectors

 The genomes of even the simplest cells are

much too large to directly analyze in detail

at the molecular level and the problem is
compounded for complex organisms.

 Human genome, contains about

6×109 base pairs (bp) in the 23 pairs of
chromosomes.
DNA Cloning with Cloning Vectors

 Cleavage of human DNA with restriction

enzymes produce about one cut for every
3000 base pairs yields (2million
fragments).
 This obstacle to obtaining pure DNA
samples from large genomes has been
overcome by recombinant DNA technology.
 With this method (Cloning) any gene can be
purified.
Recombinant DNA technology step by step

The recombinant DNA technology is the

preparation of large numbers of identical
DNA molecules (fragment)
1. DNA fragment of interest is linked through
standard 3′ → 5′ phosphodiester bonds to
a vector DNA molecule, which can replicate
when introduced into a host cell.
Recombinant DNA technology step by step

2. When a single recombinant DNA molecule,

composed of a vector plus an inserted DNA
fragment, is introduced into a host cell (competent
cell).

3. The inserted DNA is reproduced along with the

vector, producing large numbers of recombinant
DNA molecules that include the fragment of DNA
originally linked to the vector.
Function of the Cloning Vectors

The two molecules that are required for

cloning are DNA to be cloned and a cloning
vector.
Function of the Cloning vector:
1. Carries foreign DNA into a host cell.
2. Replicates inside a bacterial or yeast cell.

3. Produces many copies of itself and the

foreign DNA.
 Cloning Vectors

The vector therefore should contains

features that allow the convenient
insertion or removal of DNA fragment
in or out of the vector.
 The features of all cloning vectors

1. Small in size.

2. Sequences that permit the propagation of

itself (The replication origin) in said the

host cell (bacteria).

3. A cloning site to insert foreign DNA, good

vectors contain a site that can be cut by
many restriction enzymes.
 Multiple cloning site (MCS) site

 A multiple cloning site (MCS) contains

many restriction sites.
 These restriction sites are first cleaved
by restriction enzymes and the target
gene also, digested with the same
enzymes  then ligated into the vectors
using DNA ligase.
Multiple Cloning Site (MCS)
Multiple Cloning Site (MCS)
 The features of all cloning vectors

4. A method of selecting for bacteria or yeast

containing a vector with foreign DNA.

Usually accomplished by selectable markers for

Reporter genes or/and drug resistance .
 Reporter gene

 Reporter genes are used in some cloning

vectors to facilitate the screening of successful

clones, the features of these genes allows
successful clone to be easily identified.

 Such features present in cloning vectors may

be the lacZα fragment for α complementation

in blue-white selection.
 Reporter gene
 If the ligation was successful, the bacterial colony will
be white; if not, the colony will be blue.
 The principle of this screening is based on the ability of
β-galactosidase to produce a blue cleavage product from
an artificial chromogenic substrate, The presence of an
active β-galactosidase can be detected by X-gal, a
colourless analog of lactose that may be cleaved by β-
galactosidase.
 This results in a characteristic blue color in cells
containing a functional β-galactosidase. Blue colonies
therefore show that they may contain a vector with an
uninterrupted lacZα (therefore no insert), while white
colonies, where X-gal is not hydrolyzed, indicate the
presence of an insert in lacZα which disrupts the
formation of an active β-galactosidase.
 Introduction of a cloned DNA fragment into the
multicloning site of the vector, which is embedded in
the coding region of the LacZα gene, disrupts the
amino-terminal fragment of ß-galactosidase, which is
no longer capable of producing active β- galactosidase
.
 Selectable marker
A selectable marker is carried by the vector
to allow the selection of
positively transformed cells (cell with cloned
vector).

1. Antibiotic resistance: For example:

The beta-lactamase gene which confers
resistance to the penicillin group of beta-
lactam antibiotics like ampicillin.
 The features of all cloning vectors

5. The replication origin (ORI) is a

specific DNA sequence of 50 – 100 base pairs
that must be present in a plasmid for its
replication.
 Host-cell enzymes bind to ORI, initiating replication of the circular plasmid.

 Once DNA replication is initiated at ORI, it continues around the circular

plasmid regardless of its nucleotide sequence.

 Thus any DNA sequence inserted into such a plasmid is replicated along with
the rest of the plasmid DNA.
How can you choose the cloning Vector?

A large number of cloning vectors are

available, and choosing the vector may
depend a number of factors:

1. The size of the insert.

2. Number of Copy needed.

3. Cloning method
 How can you choose the cloning Vector?

Large insert may not be stably maintained in

a general cloning vector, especially for those
with a high copy number, therefore cloning
large fragments may require more
specialized cloning vector.
 Types of Cloning Vectors

1. Plasmid: An extra-chromosomal circular DNA

molecule that autonomously replicates inside the
bacterial cell; cloning limit to 100 to 10,000 base
pairs. or 0.1-10 kilobases (kb).

(bp↔kb 1 kb = 1000 bp)

2. Phage: Derivatives of bacteriophage lambda;

linear DNA molecules, this region can be replaced
with foreign DNA without disrupting its life cycle;
cloning limit to 8-24 kb.
 Types of Cloning Vectors

3. Cosmids: An extra-chromosomal circular

DNA molecule that combines features of
plasmids and phage; cloning limit to 35-50
kb.
4. Bacterial Artificial Chromosomes (BAC).
5. Yeast Artificial Chromosomes (YAC).
6. Human Artificial Chromosomes (HAC).
7. Mouse Artificial Chromosomes (MACs )
 Plasmids

 Plasmids are circular, double-

stranded DNA (dsDNA) molecules that are
separate from a cell’s chromosomal DNA.

 Plasmid is an autonomously replicating

circular extra-chromosomal DNA.

 Plasmids

 It occur naturally in bacteria, yeast, and some

higher eukaryotic cells, exist in a parasitic or
symbiotic relationship with their host cell.
 Plasmids range in size from a few
thousand base pairs to more than 100 kilobases
(kb).
 Like the host-cell chromosomal DNA, plasmid DNA
is duplicated before every cell division.
 Plasmids
 During cell division, at least one copy of the

plasmid DNA is segregated to each daughter

cell for assuring continued propagation of the
plasmid through successive generations of the
host cell.

 Many naturally occurring plasmids contain

genes that provide some benefit to the host

cell.
 Plasmids
 For example, some bacterial plasmids
encode enzymes that inactivate
antibiotics.

 Such drug-resistance plasmids have

become a major problem in the treatment
of a number of common bacterial
pathogens.
 Plasmids
 Many of these plasmids also contain
“transfer genes” encoding proteins that
can form a macromolecular tube,
or pilus, through which a copy of the
plasmid can be transferred to other host
cells of the same or related bacterial
species. Such transfer can result in the
rapid spread of drug-resistance plasmids.
Plasmids As A cloning vector
They are the standard cloning vectors and the
most commonly used

 The plasmids most commonly used

in recombinant DNA technology replicate
in E. coli.

 Generally, these plasmids have been

engineered (Not wild) to optimize their use
as vectors in DNA cloning.
 Plasmids As A cloning vector

 To simplify working with plasmids, their

length is reduced; many plasmid vectors are
only ≈3kb in length, which is much shorter
than in naturally occurring E. coli plasmids.
The circumference of plasmids usually is
referred to as their “length,” even though
plasmids are almost always circular DNA
molecules.
Plasmids As A cloning vector
 Many plasmids have high copy number, for
example pUC19 which has a copy number of 500-
700 copies per cell and high copy number is useful
as it produces greater yield of recombinant
plasmid for subsequent manipulation.
However low-copy-number plasmids may be preferably used in
certain circumstances, for example, when the protein from the
cloned gene is toxic to the cells.
Plasmids As A cloning vector
Bacteriophages As A cloning vector
 Bacteriophages used for cloning are
the phage λ and M13 phage.

 There is an upper limit on the amount of

DNA that can be packed into a phage (a

maximum of 25: 53 kb).

 To allow foreign DNA to be inserted into

phage, phage cloning vectors need to have

some non-essential genes deleted.
Bacteriophages As A cloning vector

 There are two kinds of λ phage vectors 

insertion vector and replacement vector.
 Insertion vectors: contain a unique cleavage site
where foreign DNA with size of 5–11 kb may be
inserted.
 In replacement vectors: the cleavage sites flank a
region containing genes not essential for the lytic
cycle, and this region may be deleted and replaced
by the DNA insert in the cloning process, and a
larger sized DNA of 8–24 kb may be inserted.
Bacteriophages As A cloning vector
Insertion vectors
Bacteriophages As A cloning vector
Replacement vectors
Difference between plasmid an bacteriophage

 Plasmid vectors replicate along with their

host cells.

 λ vectors replicate as lytic viruses, killing

the host cell and packaging the DNA into

virions.
 Cosmids as cloning vectors

 Cosmids are plasmids that incorporate a

segment of bacteriophage λ DNA that has the

cohesive end site (COS) which contains
elements required for packaging DNA into λ
head particles.

 It is normally used to clone large DNA

fragments between 28 to 45 Kb.

 Cosmids as cloning vectors

 It is a cloning vector consisting of the phage

cos site inserted into a plasmid i.e. simply

plasmid and cos sites.

 cos site is one of the cohesive, single

stranded extensions present at the ends of

the DNA molecules of certain strains of
lamda phage.
Cosmid
Cosmid Vs. Bacteriophage
 Cosmids as cloning vectors

Development of the cosmid vector is to overcome the

disadvantages of plasmids and phage vectors:-

1. The low copy number property of plasmids

overcome because of cos sites.

2. The lysis of culture by the phage virus overcome

because the vector does not have sequence for the
functional phage production.
 Artificial chromosomes

Artificial chromosomes were first assembled in budding

yeast and have since been useful in many aspects of yeast
genetics.
Several attempts have been made at building artificial
chromosomes in mammals, although these have been met
with limited success. Consequently, mini-chromosomes of
defined structure have been developed to address questions
regarding mammalian chromosome function and for
biotechnological applications.
Artificial chromosome
 Bacterial artificial chromosome

 Bacterial artificial chromosome:

Insert size of up to 350 kb can be cloned in bacterial

artificial chromosome (BAC).

BACs are maintained in E. coli with a copy number

of only one per cell.
Bacterial artificial chromosome (BAC)
 Yeast artificial chromosome

 Yeast artificial chromosome (YAC): It is genetically

engineered chromosomes derived from the DNA of

the yeast.

 Insert of up to 3,000 kb may be carried by yeast

artificial chromosome.

 This is the process that was initially used for the

Human Genome Project, however due to stability

issues, YACs were abandoned for the use
of Bacterial artificial chromosomes (BAC).
Yeast artificial chromosome
 Human artificial chromosome

 A human artificial chromosome (HAC) is a

microchromosome that can act as a new

chromosome in human cells.

 That is, instead of 46 chromosomes, the cell

could have 47 with the 47th being very

small.
 Human artificial chromosome

 Human artificial chromosome (HAC): is

potentially useful as a gene transfer vectors for gene
delivery into human cells, a tool for expression
studies and determining human chromosome
function.
 It can carry very large DNA fragment (there is no
upper limit on size for practical purposes).
 It also avoids possible insertional mutagenesis
caused by integration into host chromosomes by
viral vector.
 Human artificial chromosome

Alternative methods of creating transgenes, to

over come the problems caused by yeast
artificial chromosomes and bacterial artificial
chromosomes that lead to unpredictable
problems.
The genetic material introduced by these vectors
not only leads to different expression levels, but
the inserts also disrupt the original genome
Human artificial chromosome
 Advantages of AHCs
1. All HACs by definition contain a functional
centromere that enables the long-term stable
maintenance of HACs as single copy episomes
without integration into the host chromosomes.

2. Secondly, there is no upper size limit to DNA

cloned in a HAC, Indeed, not only single genes
but groups of genes encoding complex pathways
can be carried on a single HAC.
 Advantages of AHCs

3. Thirdly, the HACs can be transferred from

one cell to another.

4. Finally, because of the lack of viral

sequences, HAC vectors minimize adverse
host immunogenic responses and the risk of
cellular transformation.
General Steps of Cloning with Any Vector

1. Prepare the vector and DNA to be cloned by the same

restriction enzymes to generate complementary ends.

2. Ligate the foreign DNA into the vector with the

enzyme DNA ligase.

3. Introduce the cloned vector into bacterial cells by

transformation.

4. Select cells containing foreign DNA by screening for

selectable markers (usually drug resistance).
General Steps of Cloning with Any Vector
 Plasmid isolation

Plasmids may be easily isolated by cell lysis

followed by precipitation of proteins, which
traps chromosomal DNA in insoluble
fraction and after centrifugation, plasmid
DNA can be purified from soluble fraction.
 Expression vector

 Expression vector has similar features like

any vector.

 The cloned gene may be transferred from a

specialized cloning vector to an expression

vector, although it is possible to clone
directly into an expression vector.
 Elements for expression

 An expression vector must have elements

necessary for protein expression.

 These may include a strong promoter, the

correct translation initiation sequence such

as a ribosomal binding site and start codon,
a strong termination codon, and
a transcription termination sequence.
 Applications

1. Laboratory use:
Expression vector in an expression host is now the
usual method used in laboratories to produce
proteins for research.
2. Production of peptide and protein
pharmaceuticals:
Most protein pharmaceuticals are now produced
through recombinant DNA technology using
expression vectors (hormones, vaccines, antibiotics,
antibodies, and enzymes).
 Applications
3. Transgenic plant and animals:
Expression vectors have been used to introduce
specific genes in organisms,
For example in agriculture it is used to
produce transgenic plants, introduce vitamin
A precursor, beta-carotene, into rice plants, this
product is called golden rice.
Introduce a gene into plants that produces
an insecticide, which reduces the need for farmers to
apply insecticides.
 Applications
4. Transgenic animals:
 Produced to study animal biochemical processes and
human diseases.
 Green fluorescent protein is sometimes used as tags
which results in animal that can fluoresce, and this have
been exploited commercially to produce the fluorescent
GloFish.
5. Gene therapy is a promising treatment for a
number of diseases where a “Normal" gene carried by
the vector is inserted into the genome, to replace an
“Abnormal" gene or supplement the expression of
particular gene.
 Thanks a lot
with my Best Regards and My Best wishes

Amira A. AL-Hosary
E-mail: Amiraelhosary @yahoo.com
Mob. (002) 01004477501