Vectors are DNA molecules that can carry foreign DNA into another cell and also replicate it. Also known as vehicle DNA.
Essential features of a cloning vector:
- Origin of replication: ORI is a specific sequence on a DNA molecule from where replication starts. It will also replicate the foreign DNA linked to it. It also controls the copy number of the linked DNA.
- Selectable markers: these are genes present in the vector that help us in identifying and eliminating non-recombinants (non-transformants) from recombinants (transformants). E.g., Antibiotic resistance genes such as tetracycline, ampicillin, chloramphenicol or kanamycin etc., in E.coli.
- Cloning sites/ recognition sites: it is site of a restriction endonuclease that is present only once in the cloning vector
The most commonly used cloning vectors are plasmids and bacteriophages.
- Discovered by Hayes and Lederberg.
- These are extrachromosomal, self-replicating, double stranded DNA present in bacteria having an independent existence. Some have 1-2 copies/cell whereas others have 15-100 copies/cell.
- They can insert DNA of size upto15 kb.
- These are viruses infecting bacteria. Commonly used is the λ Phage & M13 Phage.
- They have very high copy numbers of their genome in bacterial cells. They are more efficient than plasmids. they can insert DNA of size 5 -11 kb.
- Formed by fusion of Plasmid with a segment of λ Phage having ‘cos’ site. They can clone large DNA fragments between 28 – 45 kb.
- They are BAC (Bacterial Artificial Chromosome) & YAC (Yeast Artificial Chromosome). Used for large DNA fragments of 300 – 3000 kb.
- The most commonly used cloning vector.
- Prepared by Boliver and Rodriguez.
- pBR 322 has antibiotic resistance gene for ampicillin (ampR) & tetracycline (tetR), are used as selectable markers.
- It has many unique restricted sites. Pvu I &Pst I in gene encoding resistance to ampR and Bam HI & Sal I in gene encoding resistance to tetR.
- It also has restricted sites for EcoR I, Cla I, Hind III, Pvu II and ‘rop’ gene. ‘rop’ (repressor of primer) codes for proteins involved in replication of the plasmid, it controls the copy number.
pBR 322 is used as a vector & Bam HI site in tetR gene is used as the unique restricted site.
- Bam HI restriction endonuclease is used to isolate the foreign DNA which is to be ligated into the vector.
- pBR 322 is also opened at the Bam HI site in the gene encoding resistance to tetR.
- Foreign DNA is ligated at the Bam HI site of tetR by a ligase enzyme.
- The recombinant plasmid loses its resistance to tetracycline due to insertion of foreign DNA.
- The recombinants are separated from non-recombinants by plating them on medium containing ampicillin.
- All Plasmids will grow on medium containing ampicillin, which is then transferred into medium containing tetracycline.
- The recombinants will not grow in it, but the non-recombinants will grow in it.
- One antibiotic resistance gene helps in selecting the non-recombinants, while the other antibiotic resistance gene gets inactivated and helps in selection of recombinants.
Alternative selectable markers:
Selective markers are developed to differentiate recombinants from non-recombinants on the basis of their ability to produce color in the presence of a chromogenic substrate.
- A recombinant DNA is created by inserting foreign DNA into the gene coding for β-galactosidases.
- This inactivates the enzyme.
- Insertional inactivation is inactivation of the gene due to insertion.
- Non-recombinants will produce blue color & recombinants do not produce any color and are marked as recombinants in the presence of chromogenic substrate.