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HotStarTaq PCR Handbook

ation of HotStarTaq DNA Polymerase and QIAGEN PCR Buffer increases specificity both at the start of and during PCR. Thus HotStarTaq DNA Polymerase is well suited to such highly sensitive PCR assays.

Nested PCR

If PCR sensitivity is too low, a nested PCR method can increase PCR product yield. Nested PCR involves two rounds of amplification reactions. The first-round PCR is performed according to the PCR Protocol using HotStarTaq DNA Polymerase. Subsequently, an aliquot of the first-round PCR product, for example 1 l of a 1-in-103104 dilution, is subjected to a second round of PCR. The second-round PCR is performed with two new primers that hybridize to sequences internal to the first-round primertarget sequences. In this way, only specific first-round PCR products (and not nonspecific products) will be
amplified in the second round. Alternatively, it is possible to use one internal and one firstround primer in the second PCR; this method is referred to as semi-nested PCR.

Single-cell PCR

HotStarTaq DNA Polymerase has been shown to successfully amplify a single-copy gene from just a single cell. The recommendations provided in Table 11 are intended to serve as a starting point for performing such a single-cell PCR from genomic template DNA. If the PCR product is undetectable or the product yield is too low, perform a nested PCR.

Table 11. Recommendations for single-cell PCR

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