PCR
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PCR is an acronym for polymerase chain reaction. It is a method for amplifying DNA in vitro.
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
- heat template/primer/dNTP/enzyme mix to 95°C for separation of DNA duplexes
- 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
- raise temperature to optimal elongation temperature of Taq or similar DNA polymerase (72-74°C)
- repeat from top 20-35 times; less cycles gives less product, too many cycles increases fraction of incomplete and erroneous products
Specific Protocols
Notes
- A discussion of the amplification efficiencies of different DNA polymerases on templates of varying length and GC content using real-time PCR [1].
References
- Arezi B, Xing W, Sorge JA, and Hogrefe HH. Amplification efficiency of thermostable DNA polymerases. Anal Biochem. 2003 Oct 15;321(2):226-35. DOI:10.1016/s0003-2697(03)00465-2 |
