2. *Genetic vectors are vehicles for delivering foreign
DNA into recipient cells.
*In molecular cloning, a vector is a DNA molecule used
as a vehicle to artificially carry foreign genetic
material into another cell, where it can be replicated
and/or expressed.
*Vectors can replicate autonomously and typically
include features to facilitate the manipulation of
DNA as well as a genetic marker for their selective
recognition.
*The cloning vectors are limited to the size of insert
that they can carry.
*Depending on the size and the application of the
insert the suitable vector is selected for a particular
purpose.
3. *Herbert Boyer, Keiichi Itakura, and Arthur Riggs
were three scientists working in the Boyer’s lab,
University of California, where they recognized a
general cloning vector.
*This cloning vector had restriction sites for cloning
foreign DNA and also, the expression of antibiotic
resistance genes for the screening of recombinant/
transformed cells.
*The first vector used for cloning purposes was
pBR322, a plasmid. It was small in size, nearly 4kB,
and had two selectable markers.
4. *
These carrier molecules should have few common features
in general such as:
*It must be self-replicating inside host cell.
*It must possess a unique restriction site for RE enzymes.
*Introduction of donor DNA fragment must not interfere
with replication property of the vector.
*It must possess some marker gene such that it can be used
for later identification of recombinant cell (usually an
antibiotic resistance gene that is absent in the host cell).
*They should be easily isolated from host cell.
5. *
Types of Cloning Vectors
A. Plasmids
B. Bacteriophage
C. Phagemids
D. Cosmids
E. Bacterial Artificial Chromosome (BAC)
F. Yeast Artificial Chromosome (YAC)
H. Retroviral vectors
6. *
1. Origin of Replication (ori)
*A specific set/ sequence of nucleotides where
replication initiates.
*For autonomous replication inside the host cell.
*Foreign DNA attached to ori also begins to replicate.
2. Cloning Site
*Point of entry or analysis for genetic engineering.
*Vector DNA at this site is digested and foreign DNA is
inserted with the aid of restriction enzymes.
*Recent works have discovered plasmids with multiple
cloning sites (MCS) which harbour up to 20 restriction
sites.
7. 3. Selectable Marker
*Gene that confers resistance to particular
antibiotics or selective agent which, under normal
conditions, is fatal for the host organism.
*Confers the host cell the property to survive and
propagate in culture medium containing the
particular antibiotics.
4. Marker or Reporter Gene
*Permits the screening of successful clones or
recombinant cells.
*Utilised extensively in blue-white selection.
8. 5. Inability to Transfer via Conjugation
*Vectors must not enable recombinant DNA to
escape to the natural population of bacterial
cells.
9. *
*Plasmids are extra chromosomal circular double stranded DNA
self-replicating elements present in bacterial cells.
*Plasmids show the size ranging from 5.0 kb to 400 kb.
Plasmids were the first vectors to be used in gene cloning.
*Plasmids are inserted into bacterial calls by a process called
transformation.
*Plasmids can accommodate an insert size of upto 10 kb DNA
fragment.
*Generally plasmid vectors carry a marker gene which is
mostly a gene for antibiotic resistance; thereby making any
cell that contains the plasmid will grow in presence of the
selectable corresponding antibiotic supplied in the media.
10.
11. *
*Bacteriophages or phages are viruses which infect
bacterial cells.
*The most common bacteriophages utilized in
gene cloning are Phage λ and M13 Phage.
*A maximum of 53 kb DNA can be packaged into
the phage.
*If the vector DNA is too small, it cannot be
packaged properly into the phage.
Examples: Phage Lambda, M13 Phage
12. *It has head, tail, and tail fibers.
*Its genome consists of 48.5 kb of DNA and 12 bp ss
DNA which comprise of sticky ends at both the
terminals.
*Since these ends are complementary, they are
cohesive and also referred to as cos sites.
*Infection by λ phage requires adsorption of tail
fibers on the cell surface, contraction of the tail,
and injection of the DNA inside the cell.
13. *
*M13 is a filamentous bacteriophage which infects E.
coli host.
The M13 genome has the following characteristics:
*Circular single-stranded DNA
*6400 base pairs long
The genome codes for a total of 10 genes (named
using Roman numerals I through X)
*These vectors are used for obtaining single-
stranded copies of the cloned DNA.
*They are utilized in DNA sequencing and in vitro
mutagenesis.
14. *M13 phages are derived from filamentous
bacteriophage M13. The genome of M13 is 6.4 kb.
*DNA inserts of large sizes can be cloned.
*From the double-stranded inserts, pure single-
stranded DNA copies are obtained.
M13 was developed into a useful cloning vector by
inserting the following elements into the genome:
*a gene for the lac repressor (lac I) protein to allow
regulation of the lac promoter
*the operator-proximal region of the lac Z gene (to
allow for a-complementation in a host with operator-
proximal deletion of the lac Z gene).
15. *a lac promoter upstream of the lac Z gene
*a polylinker (multiple cloning site) region inserted
several codons into the lac Z gene
16. *Cosmids are plasmids with a portion of bacteriophage.
*They are capable of incorporating the bacteriophage λ
DNA segment. This DNA segment contains cohesive
terminal sites (cos sites).
*Cos sites are necessary for efficient packaging of DNA
into λ phage particles.
*Large DNA fragments of size varying from 25 to 45 kb
can be cloned.
*They are also packaged into λ This permits the foreign
DNA fragment or genes to be introduced into the host
organism by the mechanism of transduction.
17. Advantages of using cosmids as vectors:
*They have high transformation efficiency and are
capable of producing a large number of clones from a
small quantity of DNA.
*Also, they can carry up to 45 kb of insert compared to
25 kb carried by plasmids and λ.
18. *
Bacterial artificial chromosomes are similar to E. coli
plasmid vectors.
*They contain ori and genes which encode ori binding
proteins. These proteins are critical for BAC replication.
*It is derived from naturally occurring F’ plasmid.
*The DNA insert size varies between 150 to 350 kb.
*BACs basically have marker like sights such as antibiotic
resistance genes and a very stable origin of replication
(ori) that promotes the distribution of plasmid after
bacterial cell division and maintaining the plasmid copy
number to one or two per cell.
19. Advantages of BACs:
*They are capable of accommodating large
sequences without any risk of rearrangement.
*BACs are frequently used for studies of genetic
or infectious disorders.
*High yield of DNA clones is obtained.
20. *
*A large DNA insert of up to 100 kb – 3000 kb can be
cloned.
*They are used for cloning inside eukaryotic cells.
These act as eukaryotic chromosomes inside the host
eukaryotic cell.
*It possesses the yeast telomere at each end.
*A yeast centromere sequence (CEN) is present which
allows proper segregation during meiosis.
*The ori is bacterial in origin.
*Both yeast and bacterial cells can be used as hosts.
21. Advantages of using YACs:
*A large amount of DNA can be cloned.
*Physical maps of large genomes like the human
genome can be constructed.
