Biology 311 Human Genetics
Cloning Vectors
What is a cloning vector?
It is a DNA molecule that carries foreign DNA into a host cell, replicates and produces
many copies of itself and the foreign DNA.
I. Features of cloning vectors
a) An origin of replication – a specific sequence at which DNA replication is initiated
b) A selectable marker- method of selecting cells containing vector with foreign
DNA from those that have not. Usually accomplished by using antibiotic resistance
markers.
c) Markers for DNA insertion- a cloning site to insert foreign DNA
II. Types of Cloning Vectors
1. Plasmids- An extrachromosomal closed-circular DNA molecule that
autonomously replicates inside bacteria cells. Has a cloning limit from
0.1 to 10 kilobases.
PROPERTIES
A) Origin of replication (ori)
Most common plasmid vector contain ori from plasmid pMB1
B) Small
This characteristic makes it easier for preparing large amounts of foreign DNA,
rapid replication and easier to purify.
C) Drug resistance gene for selective amplification
Ex: ampicillin resistance gene (ampr ) and tetracycline resistance gene (tetr) .
D. Insertion of a Polylinker
A short synthetic sequence which contains unique restriction sites for a variety
of common restriction nucleases.
E) Selection screening for recombinants
● plasmid with antibiotic resistance genes allow cells to survive on media
containing antibiotics. Ex: pBR322
● LacZ selection- this gene is interrupted in clones; in the presence of X-gal
colorless—normal lacZ releases blue pigment. If LacZ is inactive by cloned insert
white colonies are the result.
Ex: pUC18 contains all these properties.
2. Lambda phage- linear DNA molecules whose region can be replaced with
foreign DNA.
Lambda phage have a cloning capacity from 10-20 kb.
Efficiency of cloning and transformation is much higher than
in plasmids.
Lambda phage can enter host cell in two different ways:
a. Lytic cycle- virus infect host, reproduce and lyse host releasing
virus particles.
b. Lysogenic cycle- virus enters into bacteria and is replicated
along with the hosts’ DNA.
PROPERTIES
A) Removal of non-essential region
B) Genetic engineering of λ containing only a few restriction sites for a variety
of restriction enzymes.
C) Extreme ends known as COS sites
MAJOR VECTORS OF λ PHAGE
1. Replacement λ vectors- use for cloning up to 23kb in length of genomic
DNA.
2. Insertion λ vectors- use for the construction of cDNA insert into a nonessential gene (cI-gene).
3. Cosmids- combination of plasmid vector and the COS site from phage lambda
which allows foreign DNA to be inserted into the λ head.
-High transformation efficiency
-Cosmid vectors have a cloning limit capacity up to 45 kb.
Fig 1- below shows cloning by using cosmid vectors. In addition to ampr , ORI, and
polylinker as in the plasmid vector, the cosmid vector also contains a COS SITE.
(b)after vector is cleaved with restriction enzymes, they are ligated with DNA
fragments. Assembly and transformation steps are the same as cloning with λ phages.
4. BAC (Bacterial artificial chromosomes)- Based on bacterial mini-F plasmids
-Cloning capacity ≥ 300 kb
- Contains 2 genes, parA and parB which maintain copy number 1-2 per cell
- Recombinants are transferred into bacteria cells using electrical shocks these
relaxing permeability of membranes.
- Produces low yield of recombinant DNA because it contains low copy number
replicon.
- Relatively stable and resistant to rearrangement.
5. Bacteriophage P1 vector – like lambda phage, these packed their genome in a
protein coat up to a capacity of 110-115kb.
PROPERTIES AND COMPONENTS
A) Phage P1 vector system have a cloning limit capacity up to 100 kb.
B) P1 vector incorporates P1 packing site and two loxP sites.
C) Vector invades host cell which contain cre recombinase, this causes linear DNA
To be circularized. See Fig 4.15 on textbook.
6. PACs (P1-derived artificial chromosome)- a combination of P1 and F factor
system.
7. YACs (Yeast artificial chromosomes) – Capable of carrying large DNA fragments
up to 2 Mb.
- Transformation efficiency is very low
- Contains bulk of the DNA not require for
chromosome function.
ESSENTIAL COMPONENTS OF YAC VECTORS
2 Telomeres
1 centromere
1 ARS element (autonomous replicating sequence)
selective genetic markers
recognition sites for restriction enzymes
See Fig2 below – Making YACs
Fig 2- making YACs