Recombinant DNA is an artificial type of DNA produced by combining DNA of two or more species. The process of making recombinant DNA is known as molecular cloning. The basic procedure of molecular cloning includes isolating DNA, cutting DNA, joining DNA, and amplifying the recombinant DNA. The gene of interest is inserted into the vector, which serves as the carrier molecule for the gene of interest. The vector, along with the gene of interest, is referred to the recombinant DNA. The main role of restriction enzymes in gene cloning is cutting DNA. The key feature of restriction enzymes that makes them suitable for DNA manipulation is that they cut DNA at specific targets. This allows the production of desired DNA fragments for the joining purpose.
Key Areas Covered
1. What are Restriction Enzymes
– Definition, Properties, Role
2. How are Restriction Enzymes Used to Make Recombinant DNA
– Gene Cloning, Use of Restriction Enzymes in Gene Cloning
Key Terms: Cutting DNA, Gene Cloning, Gene of Interest, Molecular Cloning, Recombinant DNA Technology, Restriction Enzymes, Vector
What are Restriction Enzymes
A restriction enzyme is an endonuclease that recognizes short, specific DNA sequences known as restriction sites and cleaves the DNA at that site. They are a type of biochemical scissors produced by bacteria. Restriction enzymes defend bacteria from bacteriophages. These enzymes are isolated from bacteria and are used to cut DNA in the laboratory. The action of a restriction enzyme is shown in figure 1.
Figure 1: Action of HindIII
The ability of restriction enzymes to cut DNA at precise locations permits researchers to isolate gene-containing fragments from genomic DNA. These fragments can be inserted into vectors to produce recombinant DNA molecules.
How are Restriction Enzymes Used to Make Recombinant DNA
Recombinant DNA is a DNA molecule composed of DNA of two or more species. It mainly includes a gene of interest from a donor species and a vector that carries the gene of interest to the host cell. The main steps of the production of recombinant DNA molecules are DNA isolation, digestion with restriction enzymes, ligation of the gene of interest to the vector, and amplifying recombinant DNA molecule inside a host cell. The whole process is known as molecular cloning. Molecular cloning is shown in figure 2.
Figure 2: Molecular Cloning
The gene of interest is first isolated from biological samples in the form of genomic DNA, or it can be amplified by PCR. Sometimes, the gene of interest may be present within a vector. In order to be inserted into the vector that is suitable to carry the gene of interest into the host cell, it should be cut off from the mother molecule. As restriction enzymes precisely cut DNA by recognizing restriction sites, they can be used for this purpose. The gene of interest and the vector can be digested with the same restriction enzyme, or each end of the gene of interest can be digested by two restriction enzymes. This digestion produces compatible ends for the ligation of the gene of interest to the vector. The digestion with two restriction enzymes allows the ligation of fragments in the desired orientation. After ligation, the resultant recombinant DNA molecules are transformed into bacteria to produce a large number of copies.
Conclusion
Restriction enzymes are endonucleases that cut DNA at specific locations called restriction sites. The properties of restriction enzymes can be used to produce recombinant DNA molecules by cutting DNA at precise locations. Recombinant DNA generally contains a gene of interest inserted into a vector.
Reference:
1. “Definition of Restriction enzyme.” MedicineNet, Available here.
2. Griffiths, Anthony JF. “Making recombinant DNA.” An Introduction to Genetic Analysis. 7th edition., U.S. National Library of Medicine, 1 Jan. 1970, Available here.
Image Courtesy:
1. “HindIII Restriction site and sticky ends vector” By Helixitta – Own work (CC BY-SA 4.0) via Commons Wikimedia
2. “Construct” By Joyxi – Own work (Public Domain) via Commons Wikimedia
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