Recombinant DNA Technology

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Contents

Introduction

Recombinant DNA molecules are new artificial DNA strands that are produced by combining two unrelated (non-homologous) genes, for example, a hybrid of E. coli plasmid with human insulin gene. It is possible to join two unrelated genes from different species because all organisms in the world share the same DNA makeup (nitrogen bases, sugar, and phosphate backbone) and only differ in the sequence[1]. So one strand of DNA can complement the other strand according to Chargaff's rules. This method utilizes the transformation ability of E. coli.

Molecular Tools for making Recombinant DNA

There are severals Biological Tools required to make the Recombinant DNA:

Enzyme

Vectors:

DNA that acts as a vehicle to transport the Recombinant DNA into host cells.

A. General requirements for vector:

B. Most commonly used vectors:

DNA/mRNA

We can use either of the molecules as a source for the gene of interests.

A. DNA as the source:

B. mRNA as the source:

C. We could also use PCR to amplify particular genes of interest.

Cells

Certain types of cells are preferred as expression systems due to characteristics they have. For example yeast, insect, and mammalian cells all perform post-translation modifications required when producing human proteins. These cell types would be preferred over bacterial cells that are unable to conduct these modifications, however for simpler proteins; bacterial cells are the choice organism as they are more easily manipulated, cheaper, and they multiply rapidly.

Key Stages in the Process

Create the recombinant DNA

Cloning of recombinant DNA

Selection

Using the Recombinant DNA

Application of the Technique

Recombinant DNA is now widely used in biotechnology, medicine, research and also farming. Below are some applications of DNA recombinant Technology:

Uses In Medicine

Recombinant DNA corresponding to the A chain of human insulin is prepared and inserted into plasmids that are used to transform Escherichia coli cells. The bacteria then synthesises the Insulin chain, which is purified. A similar process is used to obtain B chains. The A and B chains are then mixed and allowed to fold and form disulphide bonds, producing active insulin molecules[3].

This technique is also applied to produce the recombinant blood clotting factor VIII for males suffering from haemophilia A[4]. This is extracted from transgenic mice milk and then purified.

This technique is also used to produce an antigen that can be used in vaccines by triggering an immune response.

This technique has also been used in the production of human erythropoietin for the treatment of anaemia and end-stage renal disease[5].

Transgenic Crops

Plants can be transformed using a plasmid from a bacterium found in soil called. Plants may be susceptible to infection, and this allows foreign DNA from the bacterium to be integrated into the plant genome[6]. This method can be used to produce transgenic crops, such as the examples below.

Transgenic Animals

RNA viruses called Retroviruses are often used as vectors to introduce foreign DNA into animal cells. Retroviruses work using reverse transcriptase to make a double-stranded DNA copy of their RNA. The virus infects the target cells, and they retain the DNA copy, producing cells that have recombinant retroviral DNA permanently inserted into their genome. This can result in an animal with an altered genotype[7].

Transformation of the germ line in mammals can also be carried out using embryonic stem cells.

Examples of transgenic animals include:

References

  1. Glick, B.R., Pasternak, J.J. and Patten, C.L. (2010) Molecular Biotechnology: Principles and Applications of Recombinant DNA, 4th edition, United States: America Society for Microbiology.
  2. Berg J., Tymoczko J. and Stryer L. (2012) Biochemistry, 7th Edition, New York: W.H. Freeman.
  3. Michael Lieberman and Allan D. Marks. (2012) Marks’ Basic Medical Biochemistry, 4th edition, Alphen aan den Rijn, Netherlands: Wolters Kluwer.
  4. Kimball, J.K., (2011) Recombinant DNA and Gene Cloning, [Online], Available: http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RecombinantDNA.html [12 Nov 2011]
  5. Winerals, Pippard, Downing, Oliver, Reid, Cotes. (1986). EFFECT OF HUMAN ERYTHROPOIETIN DERIVED FROM RECOMBINANT DNA ON THE ANAEMIA OF PATIENTS MAINTAINED BY CHRONIC HAEMODIALYSIS. The Lancet, 328(8517), 1175-1178.
  6. Hartl, D.L. and Ruvolo, M., 2012. Genetics: Analysis of Genes and Genomes. 8th ed. Jones and Bartlett Learning.
  7. Hartl, D.L. and Ruvolo, M., 2012. Genetics: Analysis of Genes and Genomes. 8th ed. Jones and Bartlett Learning.
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