A DNA molecule used as a vehicle

to transfer foreign genetic

material into another cell.
Features of vectors : origin of
replication, multiple cloning site
and selectable marker
origin of replication is a particular sequence in a
genome at which DNA synthesis/replication is initiated.
multiple cloning site, also called a polylinker, is a
short segment of DNA which contains many restriction
sites.
selectable marker is a gene introduced into a cell ,
especially a bacterium or to cells in cell culture, that
confers a trait suitable for artificial selection.

 Plasmid
Bacteriophages and other
viruses
Cosmids
Artificial chromosomes

 are found in a wide variety of bacterial

species: most plasmids have a narrow host
range
are extrachromosomal elements that
behave as accessory genetic units that
replicate and are inherited independently
have evolved a variety of mechanisms to
maintain a stable copy number in their
bacterial hosts
are dependent
contain genes coding for enzymes that are
advantageous to the bacterial host

For example, the ability to survive in normally toxic

concentrations of antibiotics such as chloramphenicol or
ampicillin is often due to the presence in the bacterium of a
plasmid carrying antibiotic resistance genes

 are double-stranded, circular, selfreplicating, extra-chromosomal DNA

molecules

 Two types of plasmid integration in a

host bacteria:

 A replicon is a genetic unit consisting

of an origin of DNA replication and its
associated control elements
A plasmid replicon can therefore be
defined as the smallest piece of
plasmid DNA that is able to replicate
autonomously and maintain normal
copy number.

 The most useful classification of naturally

occurring plasmids is based on the main
characteristic coded by the plasmid genes
Fertility or F plasmids carry only tra genes to promote
conjugal transfer of plasmids
Resistance or R plasmids carry genes conferring
resistance to antibacterial agents, such as
chloramphenicol, ampicillin
Col plasmids code for colicins, proteins that kill other
bacteria, ex. ColE1 of E. Coli
Degradative plasmids allow the host bacterium to
metabolize unusual moleculessuch as toluene and
salicylic acid, ex TOL of P. Putida
Virulence plasmids confer pathogenicity on the host
bacterium, ex. Ti plasmids of Agrobacterium
tumefaciens

 Useful properties of plasmid as cloning

vectors:
Small size
Independent origin of replication
Multiple copy number
Presence of selectable markers
The use of plasmid in cloning:
multiply (make many copies of)
express particular genes
to make large amounts of proteins
However, a plasmid can only contain inserts of
about 110 kbp

Restriction map
plasmid
pUC18/19

Multiple Cloning
Sites
pUC18

pUC19

 Advantages:
Small, easy to handle
Straightforward selection strategies
Useful for cloning small DNA fragments
(< 10kbp)
Disadvantages:
Less useful for cloning large DNA
fragments
(> 10kbp)

Cloning into a Plasmid

 Bacteriophages, or phages are viruses that

specifically infect bacteria

The structure of a typical

bacteriophage

Simplified map of the phage genome. Genes encoding

proteins required for assembly of the head and tail map
at the left end; those encoding additional proteins
required for the lytic cycle map at the right end. Some
regions of the genome can be replaced by exogenous
DNA (diagonal lines) or deleted (dotted area) without
affecting the ability of phage to infect host cells and
assemble new virions, permitting insertion of up to 25
kb of exogenous DNA between the J and N genes

 vectors
Left arm:
- head & tail
proteins

Right arm:
- DNA synthesis
- regulation
- host lysis

Deleted central
region:
- integration &
excision
- regulation

Restriction map bacteriophage vector

M13mp18

 Advantages:
Useful for cloning large DNA fragments
(10 - 23 kbp)
Inherent size selection for large inserts
Disadvantages:
Less easy to handle

 A cosmid (cos sites + plasmid) vector is a

hybrid plasmid produced by inserting the
COS sequence from phage DNA into
plasmid
COS sites are required for packing DNA into
lambda phage
A cosmid is basically a plasmid that carries
a cos site

Restriction map cosmic vector

supercos

 Advantages:
Useful for cloning very large DNA fragments
(32 - 47 kbp)
Inherent size selection for large inserts
Handle like plasmids

Disadvantages:
Not easy to handle very large DNA
fragments(~ 50 kbp)

General procedure
for cloning DNA
fragments in cosmid
vectors

1.Bacterial Artificial Chromosomes

(BACs): A bacterial artificial chromosome (BAC)
is a DNA construct based on a functional fertility
plasmid(or F-plasmid), used for transforming and
cloning in bacteria usually E. coli.
F plasmid can carry large amounts of chromosomal
DNA (over 300 kb)

BAC contains only a few genes from F, including :

- oriS and repE for replication
- sopA and sopB for keeping the copy number very low
- cat gene which confers chloramphenicol resistance
- cloning region for cloning DNA

BACs as a
cloning vector

is a vector used to clone large DNA fragments (larger than 100 kb

and up to 3000 kb). It is an artificially constructed chromosome and
contains the telomeric, centromeric and replication of origin
sequences needed for replication and preservation in yeast cells.
telomere : is a region of repetitive DNA at the ends of chromosome,
which protects the end of the chromosome from destruction
centromere : section of the chromosome required for segregation
during mitosis
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

YAC as a
cloning vector

is a microhromosome that can act as a new

chromosome in a population of human cells.
HACs behave and are constructed like
human chromosome.

Potential characteristics of human artificial

chromosomes (HACs)

Danke Schoen