2. Definition
• DNA molecule originating from a virus, a plasmid
, or the cell of a higher organism into which
another DNA fragment of appropriate size can be
integrated without loss of the vectors capacity for
self-replication; vectors introduce foreign DNA
into host cells, where it is replicated
autonomously in large quantities. Examples are
plasmids, cosmids, and yeast artificial
chromosomes; vectors are often recombinant
molecules containing DNA sequences from
several sources.
3. Characteristics of a Cloning Vector
• Origin of replication (ORI)
• This process marks autonomous replication in
vector. ORI is a specific sequence of nucleotide
in DNA from where replication starts. When
foreign DNA is linked to this sequence then
along with vector replication, foreign
(desirable) DNA also starts replicating within
host cell.
4. Selectable Marker
Besides ORI, a cloning vector should have selectable marker
gene. A selectable marker is carried by the vector to allow the
selection of positively transformed cells. Antibiotic resistance is
often used as marker, an example being the beta-
lactamase gene, which confers resistance to the penicillin group
of beta-lactam antibiotics like ampicillin. Some vectors contain
two selectable markers, for example the plasmid pACYC177 has
both ampicillin and kanamycin resistance gene
Restriction sites
It should have restriction sites, to allow cleavage of specific
sequence by specific Restriction Endonuclease. Restriction sites
in E.coli cloning vector pBR322 include HindIII , EcoRI , BamHI ,
SalI, PvuI, PstI, ClaI etc
5. Elements for expression
Main article: Expression vector
A cloning vector need not contain suitable
elements for the expression of a cloned target
gene, such as a promoter and ribosomal binding
site (RBS), many however do, and may then work
as an expression vector. The target DNA may be
inserted into a site that is under the control of a
particular promoter necessary for the expression
of the target gene in the chosen host. Where the
promoter is present, the expression of the gene is
preferably tightly controlled and inducible so that
proteins are only produced when required
6. The Major Types of Cloning Vectors
• The six major types of vectors are:
• Plasmid. Circular extrachromosomal DNA that autonomously
replicates inside the bacterial cell. Plasmids generally have a high
copy number, such as pUC19 which has a copy number of 500-
700 copies per cell.
• Phage. Linear DNA molecules derived of bacteriophage lambda.
Can be replaced with foreign DNA without disrupting its life
cycle.
• Cosmids. Another circular extrachromosomal DNA molecule that
combines features of plasmids and phage.
• Bacterial Artificial Chromosomes. Based on bacterial mini-F
plasmids.
7. Yeast Artificial Chromosomes. This is an artificial chromosome that
contains telomeres (disposable buffers at the ends of
chromosomes which are cut off during cell division) with origins of
replication, a yeast centromere (part of a chromosome that links
sister chromatids or a dyad), and a selectable marker for
identification in yeast cells.
Human Artificial Chromosome. This type of vector is potentially
useful for gene delivery into human cells, and a tool for expression
studies and determining human chromosome function. It can carry
very large DNA fragment.
All engineered vectors have an origin of replication (a replicator),
a cloning site (located where the insertion of foreign DNA neither
disrupts replication or inactivation of essential markers), and
a selectable marker (typically a gene that provides resistance to an
antibiotic.)
