Cloning is one of the most advanced scientific techniques, wherein copies of DNA fragments and cells are created by recombinant DNA technology. A vector (vehicle) is the medium which aids in cloning and the result is, amplification of the DNA molecule to produce identical copies. Thus, a cloning vector is nothing but a small stretch of DNA, originating from a plasmid, virus or a bacteriophage, into which the DNA fragment that is supposed to be cloned is inserted (it aids in the transformation process). Insertion of the foreign DNA is a complicated process and it is carried out by different tools of biotechnology, that involve, restriction and ligation enzymes. There are certain typical features of a cloning vector, that it must possess for cloning the DNA insert successfully. We shall explain you the details over here.
Note: It is important for you to know the 4 steps of cloning before understanding cloning vectors in details:
- Restriction digestion: Breaking the DNA strand with restriction enzymes.
- Ligating: Ligating the foreign strand into the vector molecule.
- Transformation: Inserting the vector DNA into the cloning vector
- Screening: Selecting the successful clones in presence of selectable markers.
Characteristics of a Cloning Vector
The cloning vector should be capable of replicating itself within the host cell to produce the clones (it's because when the foreign DNA molecule replicates itself, then clones are produced). The clones are further subjected to sorting and identification with the help of other advanced biotechnological processes. Whether you are using this for eukaryotes or for prokaryotes, there are some specific features that a cloning vector must have. They are:
ORI (Origin of Replication) Site
As the name suggests, it's the site from where the replication process initiates. Presence of this site shows that vectors are capable of autonomous replication. The foreign DNA molecule starts its replication when being tagged with the ORI sequence.
Restriction Enzyme Sites
Restriction sites are those sites, where a cut is made for the purpose of ligating the foreign DNA into the cloning vector. The sites are located at specific points on the DNA strand that are identified by specific restriction enzymes. Example: Restriction sites marked as EcoRI, stands for the restriction endonuclease that has been isolated from the bacterium E.coli , type 1 strain. The sequence of the DNA where it makes the cut is 'GAATTC' and its complementary sequence is 'CTTAAG' (a palindrome).
Presence of Selectable Markers
A selectable marker is a gene which will be specific for that vector. Presence of such genes allows artificial selection of the clones. Marker genes are also known as reporter genes and some common examples include terr and ampr. These two marker genes stand for tetracycline resistance and ampicillin resistance. The cells that have been successfully cloned (containing the foreign DNA insert) will survive in a medium incorporated with these two antibiotics.
Types of Cloning Vectors
Depending upon its ability to clone the desired number sequences and its features, a cloning vector is used for the purpose of cloning prokaryotic cells and eukaryotic cells. We have provided you examples of such vectors that are most commonly used tools of recombinant DNA technology. Take a look.
- Plasmids can carry and clone DNA stretches ranging from 8 kb -10 kb.
- Bacteriophages (Lambda phage, M13 phages) can clone up to 20 kb DNA strands.
- Cosmids have features of both phages and plasmids and they can replicate within the limit of 35-50 kb strands.
- Bacterial Artificial Chromosome (BAC) can clone 75-700 kb of DNA and is based on F (functional fertility F) plasmid.
- P1-derived Artificial Chromosome (PAC) are derived from P1 bacteriophages and they can clone DNA fragments of size ranging from 100 to 300 kb.
- Human Artificial Chromosome (HAC) or the Human Artificial Episomal Chromosome (HAEC) system, is largely used for studying gene therapies and mammalian DNA.
- Yeast Artificial Chromosome (YAC), is capable of cloning DNA fragments having base pairs ranging from 100 kb to 3000 kb. The variations of YAC are Yeast Episomal Plasmids (YEps), Yeast Integrative Plasmids (YIps) and Yeast Replicative Plasmids (YRps).