Recombinant DNA Technology

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= Introduction  =
 
= Introduction  =
  
Recombinant [[DNA|DNA]] molecules are new artificial [[DNA|DNA]] strands that are produced by combining two unrelated (non-homologous) genes, for example: hybrid of ''E. coli'' [[Plasmid|plasmid]] with human [[Insulin|insulin]] gene. It is possible to join two unrelated genes from different [[Species|species]] because all organisms in the world share the same [[DNA|DNA]] makeup&nbsp;([[Nitrogen|nitrogen]] bases, sugar, and [[Phosphate|phosphate]] backbone) and only differ in the sequence&nbsp;<ref>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.</ref> . So one strand of [[DNA|DNA]] can complement the other strand according to [[Chargaff's rules|Chargaff's rules]].  
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Recombinant [[DNA|DNA]] molecules are new artificial [[DNA|DNA]] strands that are produced by combining two unrelated (non-homologous) genes, for example: hybrid of ''E. coli'' [[Plasmid|plasmid]] with human [[Insulin|insulin]] gene. It is possible to join two unrelated genes from different [[Species|species]] because all organisms in the world share the same [[DNA|DNA]] makeup&nbsp;([[Nitrogen|nitrogen]] bases, sugar, and [[Phosphate|phosphate]] backbone) and only differ in the sequence&nbsp;<ref>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.</ref> . So one strand of [[DNA|DNA]] can complement the other strand according to [[Chargaff's rules|Chargaff's rules]]. This method utilizes the [[Transformation|transformation]] ability of ''E.coli''.&nbsp;
  
 
= Molecular Tools for making Recombinant DNA  =
 
= Molecular Tools for making Recombinant DNA  =
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== Transgenic Crop<u></u>s<br>  ==
 
== Transgenic Crop<u></u>s<br>  ==
  
Plants can be transformed using a plasmid from a bacterium found in soil called ''Agrobacterium tumefaciens. ''Plants may be sucepitble to infection and this allows foreign DNA from the bacterium to be integrated into the plant genome.<ref>Hartl, D.L. &amp;amp;amp;amp;amp; Ruvolo, M., 2012. Genetics: Analysis of Genes and Genomes. 8th ed. Jones &amp;amp;amp;amp;amp; Bartlett Learning.</ref> This method can be used to produce transgenic crops, such as the examples below.<br>  
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Plants can be transformed using a plasmid from a bacterium found in soil called ''Agrobacterium tumefaciens. ''Plants may be sucepitble to infection and this allows foreign DNA from the bacterium to be integrated into the plant genome.<ref>Hartl, D.L. &amp;amp;amp;amp;amp;amp; Ruvolo, M., 2012. Genetics: Analysis of Genes and Genomes. 8th ed. Jones &amp;amp;amp;amp;amp;amp; Bartlett Learning.</ref> This method can be used to produce transgenic crops, such as the examples below.<br>  
  
 
*<u></u>Golden rice production  
 
*<u></u>Golden rice production  
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== Transgenic Animals<br>  ==
 
== Transgenic Animals<br>  ==
  
RNA viruses called [[Retroviruses|Retroviruses]] are often used as vectors to introduce foreign DNA into animal cells. Retroviruses work using [[Reverse transcriptase|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.<ref>Hartl, D.L. &amp;amp;amp; Ruvolo, M., 2012. Genetics: Analysis of Genes and Genomes. 8th ed. Jones &amp;amp;amp; Bartlett Learning.</ref><br>  
+
RNA viruses called [[Retroviruses|Retroviruses]] are often used as vectors to introduce foreign DNA into animal cells. Retroviruses work using [[Reverse transcriptase|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.<ref>Hartl, D.L. &amp;amp;amp;amp; Ruvolo, M., 2012. Genetics: Analysis of Genes and Genomes. 8th ed. Jones &amp;amp;amp;amp; Bartlett Learning.</ref><br>  
  
 
Transformation of the germ line in mammals can also be carried out using [[Pluripotent embryonic stem cells|embryonic stem cells]].  
 
Transformation of the germ line in mammals can also be carried out using [[Pluripotent embryonic stem cells|embryonic stem cells]].  

Revision as of 10:55, 28 November 2014

Contents

Introduction

Recombinant DNA molecules are new artificial DNA strands that are produced by combining two unrelated (non-homologous) genes, for example: 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:

1. Enzyme

2. Vectors:

DNA that act as vechicle to transport the Recombinant DNA into host cells.

A. General requirements for vector:

B. Most commonly used vectors:

3. DNA/mRNA

We can use either of the molecules as source for the gene of interests.
A. DNA as source:

B. mRNA as source:

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

4. 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

1. Create the recombinant DNA

2. Cloning of recombinant DNA

3. Selection

4. Using the Recombinant DNA     

Application of the Technique

Recombinant DNA is now widely used in biotechnology, medicine, research and also farming. 
Below are several application 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 antigen that can be used in vaccines by triggering an immune response.

Transgenic Crops

Plants can be transformed using a plasmid from a bacterium found in soil called Agrobacterium tumefaciens. Plants may be sucepitble to infection and this allows foreign DNA from the bacterium to be integrated into the plant genome.[5] 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.[6]

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. Hartl, D.L. &amp;amp;amp;amp;amp; Ruvolo, M., 2012. Genetics: Analysis of Genes and Genomes. 8th ed. Jones &amp;amp;amp;amp;amp; Bartlett Learning.
  6. Hartl, D.L. &amp;amp;amp; Ruvolo, M., 2012. Genetics: Analysis of Genes and Genomes. 8th ed. Jones &amp;amp;amp; Bartlett Learning.
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