Vector for gene cloning:
A vector is the DNA molecule in which gene of interest is inserted for the purpose to construct recombinant DNA.it also act as an vehicle which is used for the transport of gene of interest into the host organism.
Different examples of vectors:
following are some vectors used for this technology
- cosmids
- phage
- plasmids
- BACs
- YACs
Qualities of a vector:
- vector has the ability to self replicate due to the presence of the origin of replication.
- vector have to replicate a lot of time per cell in order to give rise large number of copies of vectors
- a vector should must have restriction sites for restriction enzymes that will be able to accept gene of interest that is the part of different DNA cut by different restriction enzyme.
- vectors are commonly of small size that why able to easily manipulate.
- vector must have more than one suitable markers.
- vectors can easily introduced into host cell.
Choice of vector:
- choice of the vector depends on the nature of protocol or experiment in the lab.
- choice of vector also depends on the length of DNA molecules.
- it also depends on the host cell (prokaryotes & eukaryotes) to accommodate recombinant DNA.
- vectors also have promoter region which enables control expression of the inserted gene in transgenic and transfected organism.
Kind of vector:
- cloning vector
- expression vector
Cloning vector:
- cloning vector is actually a DNA molecule that have the gene of interest is inserted it transport this gene into the host cell.
- the function of cloning vector is to make a lot of number of copies of the inserted gene of interest.
- qualities of vector like presence of origin of replication, presence of more than one suitable marker, presence of unique restriction sites, facilitate in the process of cloning.
Expression vector:
- these are the cloning vector which have special expression signals which causes effective and maximum expression of the gene.
the following are the expression signals which are introduced into the gene cloned vector in order to get maximum expression.
- signals include insertion of the strong promotor region.
- and the insertion of strong termination codon.
- proper adjustment of the distance between the promoter region and cloned gene.
- it also include insertion of transcription termination sequence.
- and signals also include the insertion of the strong initiation sequence for translation .
Plasmid vector:
- plasmids are the extra circular chromosomal DNA in bacteria which is closed and double stranded in structure and self replicating organelle.
- and have drug resistance genes for bacteria
- and have origin of replication.
- the vectors that are commonly used in genetics are pBR322 and pUC18
- these are derived vectors from the natural plasmids and the both vectors are modified in such a way as are convenient for use in genetics.
pBR322:
- it is the basis of the most engineered plasmid.
- the name of the plasmid pBR322 is according to the nomenclature of the plasmid.
- the letter “p” indicates that it is a plasmid.
- the letter “BR” indicates the laboratory in which this plasmid is synthesized , BR stands for Bolivar and Rodriguez, these are researchers who constructed them.
- the number 322 distinguish this plasmid from others that are constructed in the same laboratory.
- its size is 4362 base pair.
- it has two genes that are drug-resistance genes and act as selectable marker. these genes are tetR and ampR . both these genes have special restriction site that are useful for cloning.
- the plasmid has restriction sites for restriction enzymes i.e. BamH1, HindIII, SalI, PstI, and EcoRI.
- the first three restriction sites lies in the region of tetR drug resistance gene and the fourth one(PstI) lies of ampR drug resistance gene. and EcoRI don’t lie in the region of any gene.
- gene of interest can be inserted in to any of these gene resistance regions which in next can be used to select recombinant marker after process of transformation.
- plasmid can only replicate in E. coli cell and able here to generate high number of copies.
Bacteriophage vector:
- bacteriophage or phages are called as bacterial viruses that infect this host which is bacteria by attaching to the surface of bacteria through the specific protein receptors that are present on the surface of the host.
- these bacteriophages have the ability to transfer the DNA genome of the phage into the host in which it is causing infection this idea creates the thought to design it as a vector.
- bacteriophages have heads and tails as major structural components.
- its genome is about to 48.5 kbp of the DNA molecule, which is packed into the head as a linear double stranded molecule which is with single stranded complementary terminal region of 12 nucleotides in length present at both ends (cohesive terminal i.e. 5′ GGGCGGCGACCT) which is called cos site.
- in host which is bacteria these cohesive terminals associate by pairing of base to form molecule of circular DNA and the nick are sealed.
vector sizes and insert DNA sizes that can be accommodated:
http://feeds.feedburner.com/ecarepk