PCR

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

Contents

Overview

  • Design primers
  • Prepare template
  • Prepare PCR mix
  • Run PCR cycler program
  • Analyse by gel electrophoresis

Designing primers

Designing suitable primers might be the most crucial step in PCR. This is especially true when using genomic DNA as the template. Traditionally, primers were designed using empirical guidelines. 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. Additionally, software programs automate the use empirical guidelines for primer design. See here for more details...

The general 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

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  1. Error fetching PMID 14511688: [Arezi-AnalBiochem-2003]
  2. Error fetching PMID 2999980: [Saiki-Science-1985]
    original paper on PCR

  3. Error fetching PMID 3472723: [Mullis-1986]
    first public presentation on PCR

  4. Error fetching PMID 2027781: [Yap1991]
  5. Error fetching PMID 8268790: [Douglas]
All Medline abstracts: PubMed HubMed

See also

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