An Introduction
The cloning vector can be the small piece of the DNA in which the far-off DNA is inserted for cloning. The vector might be the DNA molecule that doesn’t carry the far-off DNA in the host cell. It has the capability of self-replication and integration in the host cell. The vectors have played a crucial role in the analysis of the molecular structure of the DNA. Vectors often tend to be plasmid from a bacterium, cells from the upper organism, or the DNA from the epidemic. The target DNA gets inserted at precise sites of the vector and ligated by the DNA ligase. Next, the vector is changed into a host cell for replacement.
The Features of the Cloning Vectors
The features of the cloning vectors are as follows.
The cloning vector must possess the origin of replication so that it can self-replicate within the host cell.
It should be confined for insertion of target DNA.
It must have a marker that is selectable with antibiotic resistance gene which helps in screening recombinant organisms.
It must be small in size so that it can be easily integrated within the host cell.
It must be capable of inserting the outsized segment of the DNA.
It must possess multiple cloning sites.
It must work under eukaryotic and prokaryotic systems.
The Different Types of the Cloning Vectors
Some of the different types of cloning vectors are Plasmids, Bacteriophage, Phagemids, bacterial artificial chromosomes, and others. We will take a brief look at some of the types of cloning vectors.
Plasmids
These were the main vectors that were used in gene cloning.
These vectors are found in eukaryotes, archaea, and bacteria.
These are extrachromosomal, self-replicating, natural DNA molecules.
These vectors possess antibiotic-resistant genes with high copy numbers.
They encode the proteins that are essential for their self-replication.
pBR322, F-plasmid, pUC18 are some of the instances of the plasmid vectors.
Bacteriophage
These vectors are better than the plasmids for the cloning of large DNA insertions.
Phage λ and the M13 phage are the most common types of bacteriophages in gene cloning.
53 kb DNA is often packed in the bacteriophage.
Screening of the phage plaques is easier than screening the recombinant bacterial colonies.
Conclusion
The cloning vectors are the small pieces for inserting the foreign DNA into another cell and then making numerous copies for the equivalent. The foreign DNA gets expressed and replaced using host cell machinery. It amplifies a single copy of the DNA into several copies.
FAQs on Cloning Vector
1. What is a cloning vector in simple terms?
Think of a cloning vector as a small DNA molecule that acts like a delivery vehicle. Scientists insert a foreign piece of DNA into this vector, which then carries it into a host cell (like a bacterium). Once inside, the vector and its inserted DNA can be copied many times as the host cell divides.
2. What are the essential features that a DNA molecule needs to act as a cloning vector?
For a DNA molecule to work as a good cloning vector, it must have three key features:
- Origin of Replication (ori): This is a specific DNA sequence where replication begins, allowing the vector to make copies of itself inside the host cell.
- Selectable Marker: This is usually a gene that provides resistance to an antibiotic. It helps scientists identify which host cells have successfully taken up the vector.
- Cloning Sites (or Recognition Sites): These are unique DNA sequences recognised by restriction enzymes, where the foreign DNA can be inserted without disrupting the vector's essential functions.
3. What are the main types of cloning vectors used in biotechnology?
The most common types of cloning vectors are chosen based on the size of the DNA insert they need to carry. These include:
- Plasmids: Small, circular DNA molecules found in bacteria, ideal for cloning small DNA fragments.
- Bacteriophages: Viruses that infect bacteria. They can carry larger DNA inserts than plasmids.
- Bacterial Artificial Chromosomes (BACs): Artificially created vectors that can hold very large DNA fragments.
- Yeast Artificial Chromosomes (YACs): Used to clone extremely large DNA fragments in yeast cells.
4. Why are plasmids, like pBR322, so commonly used as cloning vectors?
Plasmids are popular cloning vectors because they are easy to handle and have all the necessary features. For example, the well-known plasmid pBR322 was one of the first vectors to be widely used. It has an origin of replication, two different selectable markers (for ampicillin and tetracycline resistance), and several unique restriction sites, making it a very effective and versatile tool for gene cloning.
5. How does a selectable marker actually help scientists?
A selectable marker helps scientists filter out the cells that did not successfully take in the cloning vector. For example, if a vector carries a gene for ampicillin resistance, researchers can grow the host bacteria on a medium containing ampicillin. Only the bacteria that have the vector (and are therefore resistant) will survive. This makes it easy to select the transformed cells for further study.
6. What is the main difference between a cloning vector and an expression vector?
The primary difference is their purpose. A cloning vector is designed simply to carry and make many copies of a piece of DNA inside a host. An expression vector goes one step further; it not only carries the DNA but also contains the necessary signals to make the host cell 'read' the gene and produce the corresponding protein. In short, a cloning vector copies DNA, while an expression vector makes protein from that DNA.
7. How are cloning vectors used in a real-world application like making human insulin?
To produce insulin, scientists first insert the human insulin gene into a plasmid (a cloning vector). This recombinant plasmid is then introduced into bacteria. The bacteria, which now carry the instructions for making insulin, are grown in large fermentation tanks. As the bacteria multiply, they not only copy the plasmid but also use it to produce large quantities of human insulin, which can then be purified and used by people with diabetes.
