Cloning Vector

By Mihir Kapse | 5 September 2024

A cloning vector is like a transportation vehicle. It helps to put foreign DNA into a microbe, mainly for cloning (that is, replication). A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. However, it does not perform cloning on its own -- it requires the DNA replication machinery of the host. It just acts as a carrier.

In 1973, Cohen and Boyer made a significant breakthrough by developing the first cloning vector, a plasmid (aA DNA molecule generally present in bacteria that can replicate independently of the genomic DNA).

plasmid

First, a fragment of DNA from a foreign microbe is cut using a restriction enzyme. The foreign DNA fragment is then joined with the vector DNA through the process of molecular ligation, often using the enzyme DNA ligase. The recombinant vector (foreign DNA + vector DNA) is then introduced into the host cell. If the host cell accepts the recombinant DNA, the replication process of DNA starts producing multiple clones of the foreign DNA with itself. The cells that successfully performed cloning are screened and separated from the group.

A cloning vector has some key features:

  1. Cloning Site: Every cloning vector has few to many sites for foreign DNA to be conveniently inserted into them.
  2. Selectable Marker: Cloning vectors, when processed through molecular ligation, may or may not get ligated. It's a desired feature that cloning vectors have a selectable marker, so that later on they can be separated from the ones that are not ligated.
  3. Reporter gene: The reporter gene is not present in all cloning vectors. They are helpful in the process of screening cells that have successfully performed cloning from the entire batch.

In 1983, Jack Szostak and Andrew Murray opened new doors by developing artificial chromosomes (particularly, Yeast Artificial Chromosomes (YAC)). Later on in the mid-late 1990s, Human Artificial Chromosomes (HAC) were developed. Artificial chromosomes are special because they can be used for cloning large fragments of DNA (up to 1 million pairs).

Presently, we know a large number of cloning vectors. However, not all can be used for a specific process. A cloning vector is chosen based on various factors: size of the insert, the copy number (number of copies of the plasmid present per cell, which determines how much the gene will be replicated) and cloning method. Some of the common cloning vectors we work with are bacteriophages, plasmids, cosmids, and artificial chromosomes.

Some of the important applications of cloning vectors are in:

  1. Gene Cloning
  2. Gene Expression Studies
  3. Genetic engineering
  4. DNA sequencing (YAC for Human Genome Project)
  5. Vaccine development

References:

  1. A Preston, Choosing a cloning vector, Humana Press, 2003
  2. Kouprina N, Earnshaw WC, Masumoto H, Larionov V, A new generation of human artificial chromosomes for functional genomics and gene therapy, Cellular and Molecular Life Sciences, April 2013

Back