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 ([[Homologous|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 ([[Nitrogen|nitrogen]] bases, sugar, and [[Phosphate|phosphate]] backbone) and only differ in the sequence<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''.  
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Recombinant [[DNA|DNA]] molecules are new artificial [[DNA|DNA]] strands that are produced by combining two unrelated ([[Homologous|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 ([[Nitrogen|nitrogen]] bases, sugar, and [[Phosphate|phosphate]] backbone) and only differ in the sequence<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|E. coli]]''.  
  
 
= Molecular Tools for making Recombinant DNA  =
 
= Molecular Tools for making Recombinant DNA  =
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== [[Vector|Vectors]]:  ==
 
== [[Vector|Vectors]]:  ==
  
DNA that acts as vehicle to transport the Recombinant DNA into host cells.  
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DNA that acts as a vehicle to transport the Recombinant DNA into host cells.  
  
 
A. General requirements for vector:  
 
A. General requirements for vector:  
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*Contain efficient origin of replication.  
 
*Contain efficient origin of replication.  
 
*Can be introduced easily to the host cells.  
 
*Can be introduced easily to the host cells.  
*Contain genes that allow for selection, such as: antibiotic resistance.  
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*Contain genes that allow for selection, such as antibiotic resistance.  
 
*May contain Expression factors.
 
*May contain Expression factors.
  
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*mRNA molecule is transcribed back to DNA using reverse transcriptase.  
 
*mRNA molecule is transcribed back to DNA using reverse transcriptase.  
*the cDNA is then being refer to [[CDNA|cDNA library]].the advantages of using cDNA is that there is no longer any intron in the DNA, so we won't produced truncated proteins.
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*the cDNA is then referring to [[CDNA|cDNA library]]. The advantages of using cDNA is that there is no longer any intron in the DNA, so we won't produce truncated proteins.
  
 
C. We could also use [[PCR|PCR]] to amplify particular genes of interest.  
 
C. We could also use [[PCR|PCR]] to amplify particular genes of interest.  
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*Model organisms are exploited in these technologies to amplifying the vector and can also be manipulated to express the product of the recombinant gene.  
 
*Model organisms are exploited in these technologies to amplifying the vector and can also be manipulated to express the product of the recombinant gene.  
*Type of cells depend on the purpose of the experiment, but most common cell type:  
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*Type of cells depends on the purpose of the experiment, but most common cell type:  
 
**[[Bacteria|Bacteria]]  
 
**[[Bacteria|Bacteria]]  
 
**[[Yeast|Yeast]]  
 
**[[Yeast|Yeast]]  
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= Application of the Technique  =
 
= 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:  
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Recombinant DNA is now widely used in biotechnology, medicine, research and also farming. Below are some applications of DNA recombinant Technology:  
  
== <u></u>Uses In Medicine<u></u> ==
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== Uses In Medicine  ==
  
 
Recombinant DNA corresponding to the A chain of human [[Insulin|insulin]] is prepared and inserted into plasmids that are used to transform ''Escherichia coli ''cells. The bacteria then synthesises the [[Insulin|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|insulin]] molecules<ref>Michael Lieberman and Allan D. Marks. (2012) Marks’ Basic Medical Biochemistry, 4th edition, Alphen aan den Rijn, Netherlands: Wolters Kluwer.</ref>.  
 
Recombinant DNA corresponding to the A chain of human [[Insulin|insulin]] is prepared and inserted into plasmids that are used to transform ''Escherichia coli ''cells. The bacteria then synthesises the [[Insulin|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|insulin]] molecules<ref>Michael Lieberman and Allan D. Marks. (2012) Marks’ Basic Medical Biochemistry, 4th edition, Alphen aan den Rijn, Netherlands: Wolters Kluwer.</ref>.  
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This technique is also used to produce an antigen that can be used in vaccines by triggering an immune response.  
 
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.<ref>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.</ref>  
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This technique has also been used in the production of human erythropoietin for the treatment of anaemia and end-stage renal disease<ref>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.</ref>.
  
== Transgenic Crop<u></u>s ==
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== 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<ref>Hartl, D.L. and Ruvolo, M., 2012. Genetics: Analysis of Genes and Genomes. 8th ed. Jones and Bartlett Learning.</ref>.This method can be used to produce transgenic crops, such as the examples below.  
 
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<ref>Hartl, D.L. and Ruvolo, M., 2012. Genetics: Analysis of Genes and Genomes. 8th ed. Jones and Bartlett Learning.</ref>.This method can be used to produce transgenic crops, such as the examples below.  
  
*<u></u>Golden rice production  
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*Golden rice production  
 
*Insect resistance crop  
 
*Insect resistance crop  
 
*Herbicide resistance crop
 
*Herbicide resistance crop

Revision as of 13:05, 4 December 2018

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:

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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