Cloning Vector: Features, Types and Gene Cloning

A cloning vector refers to a small DNA molecule in which a foreign DNA is inserted for replicating and cloning purposes. A cloning vector is a DNA molecule that is used to transport a foreign DNA molecule into a host cell. It has the ability to self-replicate and integrate into the host cell. The study of the molecular structure of DNA has been made easy by these vectors.

• The cloning vector has the capability of self-replication and can be integrated in the host cell.
• The target DNA is inserted at specific vector sites and joined by the DNA ligase. 
• Cloning vector is an important part of Gene cloning.
• Cloning vectors are widely used in genetic engineering, biotechnology, and other areas of molecular biology research.

Also Read: Chromosomes and Genes

Key Terms: DNA, Replication, Cell, Vector, Plasmid, Transformation, Gene cloning, Gene, Enzyme, Biotechnology, Molecular biology, Genetic engineering, DNA ligase

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What is a Cloning Vector?

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A cloning vector is a small piece of DNA into which a piece of foreign DNA is inserted for cloning. A vector could be a DNA molecule that prevents foreign DNA from entering the host cell.

• It has the ability to self-replicate and integrate into the host cell. 
• A cloning vector receives target DNA and then replicate it autonomously, increasing the number of copies.
• A plasmid from the bacterium is a common example of a vector.
• The target DNA is joint by DNA ligase and placed into the vector's exact locations.
• After that, the vector is inserted into the host cell for the process of transformation. 
• Plasmids, bacteriophages, and artificial chromosomes are some common types of cloning vectors. 
• Mulitple copies of a DNA fragment can be created for further analysis through a cloning vector.

Cloning Vector

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Features Of Cloning Vector

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Cloning vectors have specific features that allow them to be used for the cloning of foreign DNA fragments. Some features of cloning vectors include:

• Cloning vectors must not be too large in size allowing for easy transfer of the vector between cells.
• It should be stable within the host cell, capable of maintaining the foreign DNA fragment during replication. 
• It should be capable of working under the prokaryotic and eukaryotic systems.
• Restriction sites should be present to allow particular sequences to be broken up by restriction endonuclease.
• Cloning vectors should be compatible with the type of host cell to ensure efficient replication of the DNA fragment.
• The exact sequence of nucleotides in a DNA that acts as the origin of the replication process is the origin of replication (ORI). When foreign DNA is attached to this sequence, it begins reproducing independently within the host cell.
• A selectable marker gene such as antibiotic resistance genes, is required in the cloning vector because it enables the identification of host cells that carry the recombinant DNA.
• A cloning vector should have a specific region where the insertion of foreign DNA can occur. This region is called a multiple cloning site (MCS) or a polylinker.

With these features, cloning vectors can be efficiently used to clone foreign DNA fragments for research applications.

Also Read: Vector Borne Diseases

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Types Of Cloning Vector 

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The differnet types of cloning vectors are as follows:

Plasmids

Plasmids were the first vectors that were used in gene cloning.

• Bacteria, eukaryotes, and archaea are the common sites of these vectors.
• These are self-replicating, extrachromosomal and natural DNA molecules.
• They have a large number of copies and genes that make them resistant to antibiotics.
• They produce proteins that are required for self-replication.
• Plasmid vectors include pBR322, pUC18, and F-plasmid.

Plasmids

Bacteriophage

Bacteriophage are more efficient than plasmids for the purpose of cloning of large DNA inserts.

• In gene cloning, bacteriophages such as Phage and M13 phage are commonly used.
• In the bacteriophage, 53 kb DNA can be packed.
• It is significantly easier to screen phage plaques than to screen recombinant bacterial colonies.
• Bacteriophage is a type of cloning vector, which is a virus that infects bacteria.
• Bacteriophages can infect a broad range of bacterial hosts and can be easily propagated in large quantities.
• Bacteriophage can be used to deliver DNA into bacterial cells, which can be used for genetic modification or genetic engineering.

Structure of Bacteriophage

Phagemids

Phagemids are artificial vectors.

• They are employed in combination with the M13 phage.
• They have several cloning sites as well as an inducible lac promoter.
• Blue-white screening is used to identify them.
• Phagemids combine the features of plasmids and bacteriophages (viruses that infect bacteria).

Phagemids

Bacterial Artificial Chromosomes

Bacterial Artificial Chromosomes (BACs) are a type of cloning vector that are similar to E.coli plasmids.

• It's made from naturally produced F' plasmid.
• These are utilised in the research of genetic abnormalities.
• Large DNA sequences can be accommodated without harm.
• BACs are derived from the F-factor plasmid of E. coli, which is a naturally occurring plasmid that can replicate independently of the host chromosome.
• BACs contain a selectable marker, multiple cloning sites, and an origin of replication that allows for the replication of the plasmid in bacterial cells

Bacterial Artificial Chromosomes

Other Cloning Vectors

• Artificial Yeast Chromosomes
• Retroviral Vectors
• Cosmids
• Artificial Human Chromosomes

Also Read: Biotechnology Principles and Processes Important Questions

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Cloning Vector Gene Cloning 

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A cloning vector is a DNA molecule that is used to clone a foreign DNA fragment in a host organism.Gene Cloning is mostly utilised in DNA sequencing research.

• Gene cloning involves the isolation and amplification of a specific DNA fragment and the insertion of this fragment into a cloning vector.
• The resulting recombinant DNA molecule can then be introduced into a host cell, where it can replicate and express the cloned DNA.
• It is also used in gene therapy to treat disorders.
• In the agricultural industry, it is utilised to develop novel seed strains and enhanced fruit and vegetable varieties.
• In the pharmaceutical industry, bacteria with cloned plasmids are employed to produce vitamins and antibiotics.

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Steps Involved in Gene Cloning

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Gene cloning involves the isolation of genes from a donor cell, transferring them to a tiny carrier molecule called a vector and then replicating them in the host cell.

The steps involved are as follows:

1. Isolating the donor DNA fragment or gene

 The DNA fragment is isolated using methods such as restriction enzymes or PCR. The DNA fragment can be extracted by two methods

• The restriction endonuclease enzyme, which has a specific restriction site for its action, is used in the first method. ‘
• The reverse transcriptase enzyme is used in the second method.

2. Selecting a specific vector

A gene that has been isolated is incapable of self-replication. As a result, a specific vector with features such as selectable marker, multiple cloning sites, and an origin of replication must be chosen before insertion into the host. This vector's primary purpose is to aid in the replication process. 

3. Cutting of DNA fragment

The isolated DNA fragment is cut using the restriction enzyme to prepare the cloning vector. In the presence of DNA ligase, the vector and donor gene are combined, and a recombinant vector is generated in the mixture.

4. Ligation

The DNA fragment is ligated into the cloning vector at multiple cloning site. The ligation reaction creates a recombinant plasmid. The host organism is grown in the presence of an antibiotic that kills non-transformed cells, or contains a substrate that can only be utilized by transformed cells.

5. Transforming the recombinant vector to a host cell

The resulting recombinant vector is introduced into a host cell, bacteria or yeast, by transformation. Some other bacterias are not capable of natural transformation which further necessitates the use of an artificial technique.

5. Isolating the recombinant cell

Verification of the identity of the cloned DNA fragment can be confirmed through restriction enzyme digestion or sequencing. In this step, the host cell is allowed to develop in a culture medium. This could include both recombinant and non-recombinant cells. This mixture must be separated to determine which marker gene will be utilized. The cloned DNA fragment is transcribed and translated in the host organism to produce the desired protein.

Also Read:

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Things To Remember

• A cloning vector is a small part of DNA extracted from any organism and used to insert a foreign DNA fragment for cloning.
• Plasmids and bacteriophages can reproduce in bacterial cells without relying on chromosomal DNA for regulation.
• If a foreign piece of DNA comes into contact with bacteriophage or plasmid DNA, its number gets doubled by the plasmid or bacteriophage's number.
• Cloning vectors are used to transfer foreign DNA from one cell to another and replicate it many times.
• The foreign DNA is replicated and expressed by the host cell's machinery.
• A single copy of DNA is multiplied into multiple copies.

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Previous Year Questions

1. Two microbes found to be very useful in genetic engineering are… (NEET 2006)
2. A selectable marker is used to… (NEET 2019)
3. Commonly used vectors for human genome sequencing are… (NEET 2014)
4. Biolistics (gene-gun) is suitable for… (NEET 2012)
5. During the process of isolation of DNA, chilled ethanol is added to… (NEET 2013)
6. Some of the steps involved in the production of Humulin are given below… (Karnataka Common Entrance Test)
7. First discovered restriction endonuclease that always cuts DNA… (National Eligibility Cum Entrance Test)
8. Plasmid has been used as vector because… (NEET 2000)
9. Introduction of food plants developed by genetic engineering is not desirable because… (NEET 2002)
10. A gene whose expression helps to identify transformed cell is known as… (NEET 2017)