Polymerase Chain Reaction (PCR)

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Polymerase Chain Reaction<ref>Hartl D. L., Ruvolo M. (2012), Genetics: Analysis of genes and genomes, Eight Edition, Jones &amp; Bartlett learning (Chapter 2 DNA Structure and Genetic Variation)</ref>&nbsp;(PCR) is a technique used for the [[Amplification|amplification]] and identification of [[DNA|DNA]] or [[RNA|RNA]]. Also see [[MRNA|mRNA]]&nbsp;(including [[Transcriptase|transcriptase]]). It allows scientists to produce many millions of copies of a certain DNA sequence in a couple of hours.<br>  
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Polymerase Chain Reaction<ref>Hartl D. L., Ruvolo M. (2012), Genetics: Analysis of genes and genomes, Eight Edition, Jones &amp;amp; Bartlett learning (Chapter 2 DNA Structure and Genetic Variation)</ref>&nbsp;(PCR) is a technique used for the [[Amplification|amplification]] and identification of [[DNA|DNA]] or [[RNA|RNA]]. Also see [[MRNA|mRNA]]&nbsp;(including [[Transcriptase|transcriptase]]). It allows scientists to produce many millions of copies of a certain DNA sequence in a couple of hours.<br>
  
 
[[PCR|PCR ]]has three main stages:  
 
[[PCR|PCR ]]has three main stages:  
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Typically these steps are repeated in a cycle about 30 times generating a large amount of identical DNA copies. Therefore, PCR is often used just before doing an&nbsp;[[Electrophoresis|electrophoresis]].  
 
Typically these steps are repeated in a cycle about 30 times generating a large amount of identical DNA copies. Therefore, PCR is often used just before doing an&nbsp;[[Electrophoresis|electrophoresis]].  
  
The most important part of the PCR&nbsp;reaction is the initial design of the [[Primers|primers]]. The&nbsp;[[Primers|primers]] are normally between 18 to 20&nbsp;[[Base pairs|base pairs]] in length and must be completely&nbsp;complimentary to&nbsp;the ends of the&nbsp;[[DNA|DNA]] region of interest. Included in the&nbsp;PCR&nbsp;reaction must be both the [[Forward primers|forward]] and [[Reverse primer|reverse]] [[Primer|primers]], in&nbsp;addition to [[Taq polymerase|Taq Polymerase]]&nbsp;and&nbsp;the DNA&nbsp;template.&nbsp;Alongside these substances&nbsp;the following must also be added;&nbsp;[[Magnesium Chloride|Magnesium Chloride]], free [[Nucleotides|nucleotides]] ([[DATP|dATP]], [[DCTP|dCTP]], [[DTTP|dTTP]] and [[DGTP|dGTP]]) and a [[Tris-HCl|Tris-HCl]] (pH 8.0) [[Buffer|buffer]].<br>  
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The most important part of the PCR&nbsp;reaction is the initial design of the [[Primers|primers]]. The&nbsp;[[Primers|primers]] are normally between 18 to 20&nbsp;[[Base pairs|base pairs]] in length and must be completely&nbsp;complimentary to&nbsp;the ends of the&nbsp;[[DNA|DNA]] region of interest. Included in the&nbsp;PCR&nbsp;reaction must be both the [[Forward primers|forward]] and [[Reverse primer|reverse]] [[Primer|primers]], in&nbsp;addition to [[Taq polymerase|Taq Polymerase]]&nbsp;and&nbsp;the DNA&nbsp;template.&nbsp;Alongside these substances&nbsp;the following must also be added;&nbsp;[[Magnesium Chloride|Magnesium Chloride]], free [[Nucleotides|nucleotides]] ([[DATP|dATP]], [[DCTP|dCTP]], [[DTTP|dTTP]] and [[DGTP|dGTP]]) and a [[Tris-HCl|Tris-HCl]] (pH 8.0) [[Buffer|buffer]].<br>
  
 
PCR is carried out in a [[Thermal cycler|thermal cycler]] and the [[Enzyme|enzyme]] '[[Taq Polymerase|Taq Polymerase]]' (isolated from ''[[Thermus aquaticus|Thermus aquaticus]]'')&nbsp;is&nbsp;used as it is [[Thermostable|thermostable]]&nbsp;(is not denatured by high temperatures), therefore&nbsp;is not denatured at the high temperatures. [[Pfu|Pfu]] (''[[Pyrococcus furiosus|Pyrococcus furiosus]]'') [[DNA Polymerase|DNA&nbsp;Polymerase]] can also be used as it has better thermostability than [[Taq Polymerase|Taq Polymerase]] and it possesses 3' to 5' [[Proof reading|proof reading]] activity.  
 
PCR is carried out in a [[Thermal cycler|thermal cycler]] and the [[Enzyme|enzyme]] '[[Taq Polymerase|Taq Polymerase]]' (isolated from ''[[Thermus aquaticus|Thermus aquaticus]]'')&nbsp;is&nbsp;used as it is [[Thermostable|thermostable]]&nbsp;(is not denatured by high temperatures), therefore&nbsp;is not denatured at the high temperatures. [[Pfu|Pfu]] (''[[Pyrococcus furiosus|Pyrococcus furiosus]]'') [[DNA Polymerase|DNA&nbsp;Polymerase]] can also be used as it has better thermostability than [[Taq Polymerase|Taq Polymerase]] and it possesses 3' to 5' [[Proof reading|proof reading]] activity.  

Revision as of 10:02, 18 October 2012

Polymerase Chain Reaction[1] (PCR) is a technique used for the amplification and identification of DNA or RNA. Also see mRNA (including transcriptase). It allows scientists to produce many millions of copies of a certain DNA sequence in a couple of hours.

PCR has three main stages:

1. Heat DNA to 95°C to melt the strands

2. Cool to 50 - 65°C to allow primers to anneal

3. Heat to 72°C to allow elongation

Typically these steps are repeated in a cycle about 30 times generating a large amount of identical DNA copies. Therefore, PCR is often used just before doing an electrophoresis.

The most important part of the PCR reaction is the initial design of the primers. The primers are normally between 18 to 20 base pairs in length and must be completely complimentary to the ends of the DNA region of interest. Included in the PCR reaction must be both the forward and reverse primers, in addition to Taq Polymerase and the DNA template. Alongside these substances the following must also be added; Magnesium Chloride, free nucleotides (dATP, dCTP, dTTP and dGTP) and a Tris-HCl (pH 8.0) buffer.

PCR is carried out in a thermal cycler and the enzyme 'Taq Polymerase' (isolated from Thermus aquaticus) is used as it is thermostable (is not denatured by high temperatures), therefore is not denatured at the high temperatures. Pfu (Pyrococcus furiosus) DNA Polymerase can also be used as it has better thermostability than Taq Polymerase and it possesses 3' to 5' proof reading activity.

The technique was developed by Kary Mulis in 1983 for which he was awarded the Nobel Prize in Chemistry in 1993[2].

When a PCR machine is unavailable a water bath can be used instead.

References

  1. Hartl D. L., Ruvolo M. (2012), Genetics: Analysis of genes and genomes, Eight Edition, Jones &amp; Bartlett learning (Chapter 2 DNA Structure and Genetic Variation)
  2. http://nobelprize.org/nobel_prizes/chemistry/laureates/1993/mullis-lecture.html
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