PCR

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(steps of a PCR, primer design, PCR cycle in more easily accessible list form)
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==General Information==
 
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PCR is an acronym for polymerase chain reaction.  It is a method for amplifying DNA ''in vitro''.
PCR is an acronym for polymerase chain reaction.  It is a method for amplifying DNA ''in vitro''.
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The Basic PCR protocol calls for first heating double stranded DNA at 95 degrees celcius to separate the strands. This is followed by annealing PCR primers (55 celcius) to the single stranded DNA at the 5' ends of the DNA. A specialized enzyme, TAQ polymerase, is then added to the reaction mixture at 72 celcius (TAQ's optimum temperature). TAQ polymerase will use the primers to synthesize new DNA, creating copies of the original strand. The process is repeated numerous times for large amplification results.
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== overview ==
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* choose primers
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* prepare template
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* prepare PCR mix
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* run PCR cycler programme
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* analyse by agarose gel
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==General Procedure==
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== designing primer ==
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Designing suitable primers might be the most crucial step especially in PCRs with genome as template. In the old days, scientist chose primers by eye. Nowadays, various pieces of software help to predict the best primers including algorithms to prevent mispriming, self-complementarity and primer-primer complementarity, and binding in repeat regions. A commonly used, free primer and probe design software is Primer3.
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== the PCR cycle ==
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# heat template/primer/dNTP/enzyme mix to 95°C for separation of DNA duplexes
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# lower the temperature enough for primers to anneal specifically to the template DNA (e.g. 55°C); lowering the temperature too much increases unspecific annealing
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# raise temperature to optimal elongation temperature of ''Taq'' or similar DNA polymerase (72-74°C)
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# repeat from top 20-35 times; less cycles gives less product, too many cycles increases fraction of incomplete and erroneous products
==Specific Protocols==
==Specific Protocols==
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#Arezi-AnalBiochem-2003 pmid=14511688
#Arezi-AnalBiochem-2003 pmid=14511688
</biblio>
</biblio>
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== links ==
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* [http://en.wikipedia.org/wiki/Primer_%28molecular_biology%29 Wikipedia entry PCR]
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* [http://frodo.wi.mit.edu/primer3/input.htm primer design: Primer3]
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* [http://www.idtdna.com/Scitools/Applications/Primerquest/ primer design: PrimerQuest]
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[[Category:Protocol]]
[[Category:Protocol]]
[[Category:DNA]]
[[Category:DNA]]
[[Category:In vitro]]
[[Category:In vitro]]

Revision as of 13:34, 5 December 2006

PCR is an acronym for polymerase chain reaction. It is a method for amplifying DNA in vitro.

Contents

overview

  • choose primers
  • prepare template
  • prepare PCR mix
  • run PCR cycler programme
  • analyse by agarose gel

designing primer

Designing suitable primers might be the most crucial step especially in PCRs with genome as template. In the old days, scientist chose primers by eye. Nowadays, various pieces of software help to predict the best primers including algorithms to prevent mispriming, self-complementarity and primer-primer complementarity, and binding in repeat regions. A commonly used, free primer and probe design software is Primer3.

the PCR cycle

  1. heat template/primer/dNTP/enzyme mix to 95°C for separation of DNA duplexes
  2. lower the temperature enough for primers to anneal specifically to the template DNA (e.g. 55°C); lowering the temperature too much increases unspecific annealing
  3. raise temperature to optimal elongation temperature of Taq or similar DNA polymerase (72-74°C)
  4. repeat from top 20-35 times; less cycles gives less product, too many cycles increases fraction of incomplete and erroneous products

Specific Protocols

Notes

  1. A discussion of the amplification efficiencies of different DNA polymerases on templates of varying length and GC content using real-time PCR [1].

References

  1. Arezi B, Xing W, Sorge JA, and Hogrefe HH. . pmid:14511688. PubMed HubMed [Arezi-AnalBiochem-2003]

links

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