Wednesday, August 17, 2011

PCR Optimization

In practice PCR can fail for various reasons in part due to its sensitivity to contamination causing amplification of spurious DNA products.However, a number of techniques and procedures have been developed for optimizing PCR conditions. Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants. This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products.Also, the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA ( Pierce& Wangh, 2007).

(1) Denaturing at 94-96°C. (2) Annealing at ~65°C (3) Elongation at 72°C. Four cycles are shown here. The blue lines represent the DNA template to which primers (red arrows) anneal that are extended by the DNA polymerase (light green circles), to give shorter DNA products (green lines), which themselves are used as templates as PCR progresses(wikipedia , 2009).

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