In order to make multiple copies (clone) of the gene, it has to be joined to another DNA segment having the spontaneous ability to replicate. This DNA having the ability to replicate along with the joined DNA fragment is known as a vector or cloning vehicle. A suitable vector should have following properties:
It should have an origin (ori) for replication, one or more genetic marker for selection, a cloning site at which the gene of interest can be inserted.
After joining the gene to the vector a recombinant / chimeric DNA results which can replicate only inside a suitable cell this cell is called a host. The host provides all the enzymes and factors needed for the replication. So the host must allow:
Faithful replication of the vector must allow successful introduction of vector into the host and must be convenient for selection of desired recombinants. Several vectors like plasmids, bacterial artificial chromosomes (BAG), bacteriophages, Cosmids (Hybrid of plasmid and phage), Yeast artificial chromosomes (YAC) are in use. The most common vector is plasmid along with its host, E. coli.
ADVERTISEMENTS:
Plasmids are naturally occurring small, double stranded, extra chromosomal DNA molecules found in bacteria. The natural plasmids are not suitable for cloning. They are genetically engineered to make them suitable for cloning.
These genetically engineered cloning plasmids are : A. smaller in size, B. relaxed in replication ( replicate independent of cell replication), C. have more copy number per cell, D. have one or more marker for selection and E. contain many different restriction sites ( no particular restriction site is repeated) Two such engineered plasmids are pBR.322 (p=plasmid, developed by Bolivar and Rodriguez) and pUC (plasmid developed in University of California).A PBR322 plasmid has an origin (ori) for replication, two antibiotic resistance markers (tetracycline and ampicilin) and an EcoR I restriction site between the two antibiotic resistance genes.
The ampicilin resistance gene contains the restriction sites Pvu I and Pst I. The other resistance gene contains restriction sites hind III and Sal I. The plasmid can carry a load of insert up to lokb. But for larger insert like that of the eukaryotic genes other vectors like cosmid and YAC are used.
A phage vector can carry a load up to 10-25 kb, Cosmid can take up 35-45kb and YAC can accept a gene of 200-2000kb. A shuttle vector is compatible in both eukaryotic host and prokaryotic host.
ADVERTISEMENTS:
The yeast episomal plasmid (Yep) is an example of shuttle vector. The eukaryotic genes often require processing after transcription as they contain introns. So these genes cannot be expressed in prokaryotic hosts as they lack processing machineries. For such genes eukaryotic host-vector systems are Used. Many animal viruses are used as vectors for transformation of animal cells.
Some of the most commonly used are simian virus (SV40), papilloma virus, retroviruses etc. Another alternative for eukaryotic gene is to clone the cDXA as cDXA lack the introns they do not require processing after the transcription.