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 Insert size Features λ phages Up to 20-30 kb Genome size-47 kb, efficient

packaging system, replacement vectors usually employed, used to study individual genes.

Cosmids Up to 40 kb Contains cos site of λ phage to allow packaging, propagate in E. coli as plasmids, useful for sub-cloning of DNA inserts from YAC, BAC, PAC etc.

Fosmids 35-45 kb Contains F plasmid origin of replication and λcos site, low copy number, stable.

Bacterial artificial chromosomes (BAC)

Up to 300kb Based on F- plasmid, relatively large and high capacity vectors.

P1 artificial chromosomes (PACs) Up to 300 kb 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

Yeast artificial chromosomes (YAC) Up to 2000kb Allow identification of successful transformants (BAC clones are highly stable and highly efficient)

Types of Cloning Vectors:

Vector Insert size Source Application Plasmid ≤ 15 kb Bacteria Subcloning and

downstream manipulation, cDNA cloning and expression assays

Lambda Phage 5-20 kb Bacteriophage λ Genomic DNA cloning, cDNA cloning and expression library

Cosmid 35-45 kb Plasmid containing a bacteriophage λ cos site

Genomic library construction

BAC (bacterial artificial chromosome)

75-300 kb Plasmid ocntaining ori from E.coli F- plasmid

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

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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 non- specific 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 downstream of the 3´ recognition site

Restriction and modification

Single multifunctional enzyme

Separate nuclease and methylase

Separate enzymes sharing a common subunit

Nuclease subunit structure

Heterotrimer Homodimer Heterodimer

Cofactors 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 Non-viral Vectors

Adenovirus Lipid complex

Retrovirus Liposomes

Adeno- Associated Virus Peptide/protien

Lentivirus Polymers

Vaccinia virus

Herpes simplex virus

• 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

Genome Insert capacity (kb)

Specific integration

Long-term maintenance

RNA intermediate

Retroviruses ssRNA with DNA intermediate

1-7 Y Y RNA with DNA intermediate

Adenovirus dsDNA 2-38 N N N

Adeno-associated virus

ssDNA 4.5 Y Y N

Herpes simplex virus

dsDNA 50 N Y N

Lentivirus RNA with DNA intermediate

7-18 Y Y RNA with DNA intermediate

Viral vector Insert type Insert size Immunogenicity Host genome integration

Adeno virus DNA 2-8 kb Very high Non integrating Retro virus RNA 2-8 kb Low Integrating Lentivirus RNA 7-18 kb Low Integrating Adeno associated virus (AAV)

DNA 4.5 kb Low Non integrating

Herpes simplex virus (HSV)

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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 λ 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.