Pyrosequencing

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Pyrosequencing (also known as [[454-sequencing|454-sequencing]]) is a method of [[DNA Sequencing|DNA Sequencing]]. It involves using an [[Enzyme|enzyme]] called [[Luciferase|luciferase]], amongst others, to determine the [[Nucleotide|nucleotide]] sequence based upon the light emitted when a correct [[DNTP|dNTP]] binds to a template.  
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Pyrosequencing (also known as [[454-sequencing|454-sequencing]]) is a method of [[DNA Sequencing|DNA Sequencing]].This technique allows the sequencing of DNA between 400-600Mb per hour run. It involves using an [[Enzyme|enzyme]] called [[Luciferase|luciferase]], amongst others, to determine the [[Nucleotide|nucleotide]] sequence based upon the light emitted when a correct [[DNTP|dNTP]] binds to a template.  
  
 
The 454 method of sequencing has increased the speed and efficiency of [[DNA|DNA]] sequencing. It incorporates a laminar flow method of delivering reagents and washing away unused reagents along with etched fibre optic slides to help measure the light output of the reactions occurring. It is a massively parallel process reducing the cost of sequencing greatly.   
 
The 454 method of sequencing has increased the speed and efficiency of [[DNA|DNA]] sequencing. It incorporates a laminar flow method of delivering reagents and washing away unused reagents along with etched fibre optic slides to help measure the light output of the reactions occurring. It is a massively parallel process reducing the cost of sequencing greatly.   
  
The 454 method of sequencing intially involves a [[Primer|primer]] being added to a single stranded template. This is then incubated with substrates of adenosine 5’ phosphosulfate and luciferin, as well as the enzymes of [[DNA Polymerase|DNA polymerase]], [[ATP sulphurylase|ATP sulphurylase]], luciferase and apyrase. <br>[[DNA polymerase|DNA polymerase]] adds complementary [[dioxyribonucleotide triphosphates|dioxyribonucleotide triphosphates]] (dNTP) to the template strand; each time a [[dNTP|dNTP]] is added, a [[pyrophosphate|pyrophosphate]] (PPi) is released. The released PPi is converted into [[ATP|ATP]], by a sulphurylase enzyme, which then helps to drive the conversion of luciferin to [[oxyluciferin|oxyluciferin]]. The latter conversion is mediated by luciferase and results in the production of light. <br>The light produced is automatically detected and can be visualised as peaks on a graph; the height of each peak is proportional to the number of dNTPs added&nbsp;<ref>QIAGEN, 2011. Principles of Pyrosequencing Technology. [online] Available from: http://www.pyrosequencing.com/DynPage.aspx?id=7454 [Accessed 17th November 2011]</ref>.<br>  
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Before carrying out pyrosequencing, preparation of DNA fragments require nebulisation process,where it can produce many DNA fragments at once. Subsequently, adaptors are added in order to ligate with the DNA fragments, which act as a primer site. NaOH is added into the sample to denature the DNA into single stranded strand. As a result, every DNA fragments terminated with common adaptor sequences.
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The 454 method of sequencing intially involves a [[Primer|primer]] being added to a single stranded template. This is then incubated with substrates of adenosine 5’ phosphosulfate and luciferin, as well as the enzymes of [[DNA Polymerase|DNA polymerase]], [[ATP sulphurylase|ATP sulphurylase]], luciferase and apyrase. <br>[[DNA polymerase|DNA polymerase]] adds complementary [[Dioxyribonucleotide triphosphates|dioxyribonucleotide triphosphates]] (dNTP) to the template strand; each time a [[DNTP|dNTP]] is added, a [[Pyrophosphate|pyrophosphate]] (PPi) is released. The released PPi is converted into [[ATP|ATP]], by a sulphurylase enzyme, which then helps to drive the conversion of luciferin to [[Oxyluciferin|oxyluciferin]]. The latter conversion is mediated by luciferase and results in the production of light. <br>The light produced is automatically detected and can be visualised as peaks on a graph; the height of each peak is proportional to the number of dNTPs added&nbsp;<ref>QIAGEN, 2011. Principles of Pyrosequencing Technology. [online] Available from: http://www.pyrosequencing.com/DynPage.aspx?id=7454 [Accessed 17th November 2011]</ref>.<br>  
  
 
454 sequencing has been shown to give "99.5% accuracy at 200 bases" and is capable of giving ~20 megabases of 110 base-reads taking around 8 hours and looks to increase as the system becomes more refined.  
 
454 sequencing has been shown to give "99.5% accuracy at 200 bases" and is capable of giving ~20 megabases of 110 base-reads taking around 8 hours and looks to increase as the system becomes more refined.  
  
The downsides are that the system still has a relatively low throughput. This increases the cost per base of the system&nbsp;<ref>Rothberg, J.M. &amp;amp;amp;amp;amp;amp;amp;amp; Leamon, J.H., 2008. The development and impact of 454 sequencing. Nature biotechnology, 26(10), pp.1117-24. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18846085.</ref>.&nbsp; &nbsp;  
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The downsides are that the system still has a relatively low throughput. This increases the cost per base of the system&nbsp;<ref>Rothberg, J.M. &amp;amp;amp;amp;amp;amp;amp;amp;amp; Leamon, J.H., 2008. The development and impact of 454 sequencing. Nature biotechnology, 26(10), pp.1117-24. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18846085.</ref>.&nbsp; &nbsp;  
  
 
Alternatives to this system include the [[Solexa|Solexa]]&nbsp;system&nbsp;<ref name="null">Ronaghi M. Pyrosequencing Sheds Light On DNA Sequencing, Genome Res. 2001 11: 3-11</ref>.  
 
Alternatives to this system include the [[Solexa|Solexa]]&nbsp;system&nbsp;<ref name="null">Ronaghi M. Pyrosequencing Sheds Light On DNA Sequencing, Genome Res. 2001 11: 3-11</ref>.  

Latest revision as of 15:21, 9 October 2014

Pyrosequencing (also known as 454-sequencing) is a method of DNA Sequencing.This technique allows the sequencing of DNA between 400-600Mb per hour run. It involves using an enzyme called luciferase, amongst others, to determine the nucleotide sequence based upon the light emitted when a correct dNTP binds to a template.

The 454 method of sequencing has increased the speed and efficiency of DNA sequencing. It incorporates a laminar flow method of delivering reagents and washing away unused reagents along with etched fibre optic slides to help measure the light output of the reactions occurring. It is a massively parallel process reducing the cost of sequencing greatly. 

Before carrying out pyrosequencing, preparation of DNA fragments require nebulisation process,where it can produce many DNA fragments at once. Subsequently, adaptors are added in order to ligate with the DNA fragments, which act as a primer site. NaOH is added into the sample to denature the DNA into single stranded strand. As a result, every DNA fragments terminated with common adaptor sequences.

The 454 method of sequencing intially involves a primer being added to a single stranded template. This is then incubated with substrates of adenosine 5’ phosphosulfate and luciferin, as well as the enzymes of DNA polymerase, ATP sulphurylase, luciferase and apyrase.
DNA polymerase adds complementary dioxyribonucleotide triphosphates (dNTP) to the template strand; each time a dNTP is added, a pyrophosphate (PPi) is released. The released PPi is converted into ATP, by a sulphurylase enzyme, which then helps to drive the conversion of luciferin to oxyluciferin. The latter conversion is mediated by luciferase and results in the production of light.
The light produced is automatically detected and can be visualised as peaks on a graph; the height of each peak is proportional to the number of dNTPs added [1].

454 sequencing has been shown to give "99.5% accuracy at 200 bases" and is capable of giving ~20 megabases of 110 base-reads taking around 8 hours and looks to increase as the system becomes more refined.

The downsides are that the system still has a relatively low throughput. This increases the cost per base of the system [2].   

Alternatives to this system include the Solexa system [3].

References

  1. QIAGEN, 2011. Principles of Pyrosequencing Technology. [online] Available from: http://www.pyrosequencing.com/DynPage.aspx?id=7454 [Accessed 17th November 2011]
  2. Rothberg, J.M. &amp;amp;amp;amp;amp;amp;amp;amp; Leamon, J.H., 2008. The development and impact of 454 sequencing. Nature biotechnology, 26(10), pp.1117-24. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18846085.
  3. Ronaghi M. Pyrosequencing Sheds Light On DNA Sequencing, Genome Res. 2001 11: 3-11

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