In nested PCR, two pairs of PCR primers with one set internal to the other (nested) are used to sequentially amplify a single locus. The first pair is used to amplify the locus as in any PCR assay. A dilution of the first PCR reaction then is amplified with the nested primers. Alternatively, seminested PCR is performed using one of the original PCR primers and one new internal primer in a second round of amplification. Both nested and semi-nested PCR generate a second PCR product that is shorter than the first one.21 The logic behind this strategy is that if the wrong locus was amplified incorrectly or nonspecifically, the probability is very low that it would be amplified a second time by a second pair of primers. Thus, nested PCR enhances specificity while also increasing sensitivity. The problem with nested PCR is the high risk of amplicon contamination when the first-round PCR products are used to set up the second round of PCR with the nested primers (see section below on amplicon contamination control). For this reason, many clinical laboratories do not use nested PCR procedures.
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