Difference between revisions of "PCR"
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Latest revision as of 03:29, 22 December 2011
PCR is an acronym for polymerase chain reaction. It is a method for amplifying DNA in vitro.
- Design primers
- Prepare template
- Prepare PCR mix
- Run PCR cycler program
- Analyse by gel electrophoresis
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
- 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
- A discussion of the amplification efficiencies of different DNA polymerases on templates of varying length and GC content using real-time PCR .
- Arezi B, Xing W, Sorge JA, and Hogrefe HH. Amplification efficiency of thermostable DNA polymerases. Anal Biochem. 2003 Oct 15;321(2):226-35.
- Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, and Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350-4.
original paper on PCR
- Mullis K, Faloona F, Scharf S, Saiki R, Horn G, and Erlich H. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb Symp Quant Biol. 1986;51 Pt 1:263-73.
first public presentation on PCR
- Yap EP and McGee JO. Short PCR product yields improved by lower denaturation temperatures. Nucleic Acids Res. 1991 Apr 11;19(7):1713.
- Douglas A and Atchison B. Degradation of DNA during the denaturation step of PCR. PCR Methods Appl. 1993 Oct;3(2):133-4.