Plasmids are the most commonly used cloning vectors. Plasmids are popular because they allow cloning and manipulation of small pieces of DNA thereby being helpful in molecular biology techniques. One of the first widely used plasmid DNA vectors, called pBR322 was designed to have genes for ampicillin and tetracycline resistance and several useful restriction sites.
FEATURES OF DNA CLONING VECTORS
Modern plasmid DNA cloning vectors usually include the features.
- SIZE – They should be small enough to be easily separated from chromosomal DNA of host bacteria.
- ORIGIN OF REPLICATION(ori) – The site for DNA replication that allows plasmids to replicate separately from the host cell’s chromosome. The number of plasmids in the cell is called the copy number. Most of the desirable cloning plasmids are known as high copy number plasmids because they can replicate to create hundreds or thousands of plasmid copies.
- MULTIPLE CLONING SITE – The MCS , also called a poly linker is a stretch of DNA with recognition sequences for many different restriction enzymes. A MCS provides a lot of opportunities for possible DNA inserts to be added.
- SELECTABLE MARKER GENES – These genes allow the selection and identification of bacteria that have been transformed with a recombinant plasmid compared to non transformed cells. Example, ampicillin resistance and tetracycline resistance and lacZ gene used for blue – white selection.
- RNA POLYMERASE PROMOTER SEQUENCE – These sequences are used for transcription of RNA in vivo and in vitro by RNA polymerase.
- DNA SEQUENCING PRIMER SEQUENCE – These sequences permit nucleotide sequencing of cloned DNA fragments that have been inserted into the plasmid.
TYPES OF VECTORS
Each vectors has its significant role in molecular biology techniques. Therefore, there are different cloning vectors.
- BACTERIOPHAGE VECTORS
DNA from bacteriophage lambda one of the first phage vectors used in cloning. The lambda chromosome is a linear 49 kb size structure. Cloned DNA is inserted into restriction sites in the centre of the lambda chromosome Recombinant chromosomes are hen packaged into viral particles in vitro, and these phages are used to infect E.coli growing as a lawn. At each end of the lambda chromosome are 12 nucleotide sequences called cohesive sites (COS) that can base pair with each other . When lambda infects E.coli as a host, the Lambda chromosome uses these COS sites to circularize and then replicate. A primary advantage of using these vectors is that they allow cloning of larger DNA fragments than plasmids.
2. COSMID VECTORS
Cosmid vectors contain COS ends of lambda DNA, a plasmid origin of replication, and genes have been removed. DNA is cloned into a restriction site, and the cosmid is packaged into viral particle, a s is done with bacteriophage vectors. Bacterial colonies are formed on a plate, and recombinants are screened by antibiotic selection. A primary advantage of cosmids is that they allow for the cloning of DNA fragments in the 20- 45 kb range.
3. EXPRESSION VECTORS
Protein expression vectors allow high level expression of eukaryotic proteins with in the bacterial cells because they contain a prokaryotic promoter sequence adjacent to the site where DNA is inserted into the plasmid. Bacterial RNA polymerase can bind to the promoter and synthesize large amounts of RNA which is then translated into protein. Protein can then be isolated using biochemical techniques.
4. BACTERIAL ARTIFICIAL CHROMOSOMES
Bacterial artificial chromosome (BACs) are large low copy number plasmids, present as one or two copies in bacterial cells that contain genes encoding the F- factor. BACs can accept DNA inserts in the 100 – 300 kb range. BACs were widely used in the Human Genome Project to clone and sequence large pieces of human chromosomes.
5. YEAST ARTIFICIAL CHROMOSOMES
Yeast artificial chromosomes (YACs) are small plasmids grown in E.coli and introduced into yeast cells. A YAC is a miniature version of a eukaryotic chromosome. YACs contain an origin of replication, selectable markers, two telomeres, and a centromere that allows for replication of the YAC and segregation into daughter cells during cell division. Foreign DNA fragments are cloned into the restriction site into the center of the YAC. YACs are useful for cloning large fragments of DNA from 200 kb to approx. 2 megabases (mb= 1 million bases) in size. YACs have also been used in Human Genome Project.
6. Ti VECTORS
These are naturally occurring plasmids isolated from the bacterium Agrobacterium tumefaciens. A soil borne Gram negative pathogen that infects dicot plants by causing crown gall disease. On entering a cell the A. tumefaciens inserts a apart of its DNA(T-DNA) from the Ti plasmid into the host chromosome. Therefore the plant geneticists exploited this property if the bacteria and used it to insert their gene of interest using A. tumefaciens as a vector into the host cell.