Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization

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Abstract
This unit describes a method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield. Hot-start methods are described which can greatly improve specificity, sensitivity, and yield. This protocol suggests some relatively inexpensive methods to achieve hot start, and lists several commercial hot-start options which may be more convenient, but of course more expensive. The unit has recently been updated to include new information on reagents to enhance the reaction, better cycling parameters, and innovations in robotics and high-performance thermocyclers.