The polymerase chain reaction (PCR) has rapidly become an essential tool within the diagnostic laboratory. Therefore, it is crucial when setting up a new PCR-based test to ensure that the PCR reaction is carefully designed to be as robust and reliable as possible. Usually, little optimization is required. However, there are some instances when a particular region of DNA proves difficult to amplify by PCR. A number of factors are important to consider when choosing PCR conditions, and these are discussed in this chapter.