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Vaccinia virus (VACV or VV) is a large, complex, enveloped virus belonging to the poxvirus family.

It
has a linear, double-stranded DNA genome approximately 190 kbp in length, and which encodes
approximately 250 genes

VECTORS AND THEIR INSERTING SIZE


Vector
phages

Insert size
Up to 20-30 kb

Cosmids

Up to 40 kb

Fosmids

35-45 kb

Bacterial artificial chromosomes


(BAC)
P1 artificial chromosomes (PACs)

Up to 300kb

Yeast artificial chromosomes (YAC)

Up to 2000kb

Up to 300 kb

Features
Genome size-47 kb, efficient
packaging system, replacement vectors
usually employed, used to study
individual genes.
Contains cos site of phage to allow
packaging, propagate in E. coli as
plasmids, useful for sub-cloning of
DNA inserts from YAC, BAC, PAC
etc.
Contains F plasmid origin of
replication and cos site, low copy
number, stable.
Based on F- plasmid, relatively large
and high capacity vectors.
Derived from DNA of P1
bacteriophage, combines the features
of P1 and BACs, used to clone larger
genes and in physical mapping,
chromosome walking as well as
shotgun sequencing
of complex genomes
Allow identification of successful
transformants (BAC clones are highly
stable and highly efficient)

Types of Cloning Vectors:


Vector
Plasmid

Insert size
15 kb

Source
Bacteria

Lambda Phage

5-20 kb

Bacteriophage

Cosmid

35-45 kb

BAC (bacterial
artificial
chromosome)

75-300 kb

Plasmid containing a
bacteriophage cos
site
Plasmid ocntaining
ori from E.coli Fplasmid

Application
Subcloning and
downstream
manipulation, cDNA
cloning and
expression assays
Genomic DNA
cloning, cDNA
cloning and
expression library
Genomic library
construction
Analysis of large
genomes

YAC (yeast artificial


chromosome)

100-1000 kb (1 Mb)

Saccharomyces
cerevisiae
centromere, telomere
and autonomously
replicating sequence

Analysis of large
genome, YAC
transgenic mice

MAC (mammalian
artificial
chromosome)

100 kb to > 1 Mb

Mammalian
centromere, telomere
and origin of
replication

Under development
for use in animal
biotechnology and
human gene therapy

---------------------------------------------------------------------------------------------------------------------------Classification of Restriction Endonuclease (Most widely used Type


II)
Property

Type I RE

Type II RE

Type III RE

Abundance

Less common than Type II

Most common

Rare

Recognition site

Cut both strands at a nonspecific location > 1000 bp


away from recognition site

Cut both strands at a specific,


usually palindromic
recognition site (4-8 bp)

Cleavage of one strand,


only 24-26 bp down
stream of the 3
recognition site

Restriction and
modification

Single
multifunctional
enzyme

Separate nuclease
and methylase

Separate enzymes
sharing a
common subunit

Nuclease subunit
structure
Cofactors

Heterotrimer

Homodimer

Heterodimer

ATP, Mg2+, SAM

Mg2+

Mg2+ (SAM)

DNA cleavage
requirements

Two recognition
sites in any
orientation

Single recognition
site

Two recognition
sites in a
head-to-head
orientation

Enzymatic turnover

No

Yes

Yes

DNA translocation

Yes

No

No

Site of methylation

At recognition site

At recognition site

At recognition site

Vectors used in gene therapy


Viral Vector
Adenovirus

Non-viral Vectors
Lipid complex

Retrovirus
Adeno- Associated Virus

Liposomes
Peptide/protien

Lentivirus
Vaccinia virus
Herpes simplex virus

Polymers

Direct gene transfer methods like mechanical, electroporation, gene gun are
also ued to transfer genes into target cells.

Properties of viral vectors used in gene transfer


. Viral
vectors
Retroviruses

Genome
ssRNA with
DNA
intermediate
dsDNA
ssDNA

Adenovirus
Adenoassociated
virus
Herpes
dsDNA
simplex virus
Lentivirus
RNA with
DNA
intermediate

Insert
capacity (kb)
1-7

Specific
integration
Y

Long-term
maintenance
Y

RNA intermediate

2-38
4.5

N
Y

N
Y

N
N

50

7-18

RNA with DNA intermediate

RNA with DNA intermediate

Viral vector

Insert type

Insert size

Immunogenicity

Adeno virus
Retro virus
Lentivirus
Adeno associated
virus (AAV)
Herpes simplex
virus (HSV)

DNA
RNA
RNA
DNA

2-8 kb
2-8 kb
7-18 kb
4.5 kb

Very high
Low
Low
Low

Host genome
integration
Non integrating
Integrating
Integrating
Non integrating

DNA

>30kb

Low

Non integrating

------------------------

Restriction enzymes that generate sticky ends


Genomic DNA can be digested with commonly available restriction enzymes that generate sticky ends.
For example, digestion of genomic DNA with the restriction enzyme Sau3AI (recognition sequence
5-GATC-3) generates DNA fragments that are compatible with the sticky end produced by BamHI
(recognition sequence 5-GGATCC-3) cleavage of a vector. Once the DNA fragments are produced,
they are cloned into a suitable vector.
Restriction enzymes generating blunt ends

The genomic DNA can be digested using restriction enzymes that generate blunt ends e.g. HaeIII and
AluI.
Blunt ends are converted into sticky ends prior to cloning. These blunt ended DNA fragments can be
ligated to oligonucleotides that contain the recognition sequence for a restriction enzyme called linkers
or possess an overhanging sticky end for cloning into particular restriction sites called adaptors.
Linkers
Linkers are short stretches of double stranded DNA of length 8-14 bp that have recognition site for
restriction enzymes. Linkers are ligated to blunt end DNA by ligase enzyme. The linker ligation is more
efficient as compared to blunt-end ligation of larger molecules because of the presence of high
concentration of these small molecules in the reaction. The ligated DNA can be digested with
appropriate restriction enzyme generating cohesive ends required for cloning in a vector. The restriction
sites for the enzyme used to generate cohesive ends may be present within the target DNA fragment
which may limit their use for cloning.
Adapters
These are short stretches of oligonucleotide with cohesive ends or a linker
digested with restriction enzymes prior to ligation. Addition of adaptors to the
ends of a DNA converts the blunt ends into cohesive ends
There are three approaches to make recombinant DNA:
1. Transformation
2. Non- bacterial transformation/transfection
3. Phage introduction/transduction
Transformation:
Transformation is direct uptake of exogenous DNA via cell membrane leading to
incorporation into the host DNA. It is commonly occurred in bacteria.
Transformation requires different tools of molecular biology to insert foreign DNA
into the host. For example, vector to carry the foreign DNA to the host;
restriction enzymes to cut the DNA in specific site; ligase to join two DNA
molecule etc.
Non-bacterial transformation/transfection:
The process of foreign DNA uptake by host cell driven by mechanical or chemical factors is classified
under non-bacterial transformation, also termed as transfection. Different methods of non-bacterial
transformation are microinjection, liposome mediated transformation, biolistics etc.
Phage introduction/transduction:
Phage vector is used to carry and replicate foreign DNA inside the bacterial host
system. The phage DNA inserts into the host chromosome by recombination.
Phage had short regions of single-stranded DNA with complementary base
sequences called cohesive (cos) sites. Base pairing between the complementary
cos sites allows the linear genome to form a circle within the host bacterium.
Circularized viral genome can be integrated into the bacterial genome by
homologous recombination between attP site of viral genome and attB site of
bacterial genome.