The PCR product should be a fragment or fragments of DNA of defined length. Before the PCR product is used in further applications, it should be analyzed. For diagnostic applications, this analysis can be performed on an ongoing basis for every patient specimen or during the initial method development and verification. First, reactions should be examined to ensure product is actually formed. This seems intuitive, but when amplicon is detected with a probe, unexpectedly negative results could be due to either lack of amplification or probe hybridization/detection problems. Although biochemistry is an exact science, not every PCR reaction is successful. Causes are many and include poor quality of target DNA, too much
target DNA, lack of sequence homology between primers and the intended target, and failure to optimize PCR conditions. PCR product must also be the correct size. Unexpected amplicon size indicates that the target region itself is different than expected, that the target sequence is shared, or that amplification conditions are suboptimal and allow nonspecific annealing. PCR product should also be evaluated to ensure that the correct number of distinct products are produced. In most diagnostic applications, a single amplicon is generated by one primer pair. Additional, unintended product is usually produced as a result of suboptimal amplification conditions (poor primer design, Taq or MgCl2 concentration too high, annealing temperature not optimized). PCR product is analyzed by electrophoresis in agarose gels and visualization with ethidium bromide. DNA fragment size is determined by comparison with known molecular weight markers. Agarose gel electrophoresis is also a PCR detection format but is not recommended as a stand-alone method, as amplicon sequence cannot be confirmed. PCR detection formats are discussed in detail in another chapter of this text.
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