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prostar RT-PCR Systems for Robust High-Fidelity RNA Amplification

they require additional cumbersome manipulations (e.g., buffer additions and the use of wax barriers).

RT-PCR Accuracy Is Influenced by DNA Polymerase Fidelity

The frequency of mutations in RT-PCR products is thought to be influenced by the replication fidelity of both the RT and the DNA polymerase, under the particular reaction conditions employed (e.g., number of duplications, pH, and [Mg2+]). The mutation frequency (M.F.) of an RNA amplification procedure can be estimated from the RT error rate, the DNA polymerase error rate, the number of cDNA template duplications, and the amplicon size. For example, consider the situation when an RNA template is reverse transcribed with MMLV-RT (error rate of 3.3 x10-5/base6), and then a 1-kb portion is PCR amplified for 20 duplications (106-fold amplification) using Stratagenes PfuTurbo DNA polymerase (error rate of 1.3 x 10-6 M.F./bp/duplication7). The percentage of 1-kb cDNAs that contain errors is calculated to be 3.3% (3.3 x 10-5/base x 1000 bases), while an additional 2.6% of the final clones would contain mutations introduced by PfuTurbo DNA polymerase [(1.3 x 10-6)(1000 bp)(20 duplications)]. In this example, the overall M.F. would be 5.9%. In contrast, if the same RT-PCR reaction was carried out with AMV-RT (error rate of 5.9 x 10-5/base6) and Taq DNA polymerase (8 x 10-6 M.F./bp/duplication7), 5.9% of 1-kb cDNAs would contain RT-generated errors, and an additional 16% of the final clones would contain Taq-generated mutations [(8.0 x 10-6) x (1000 bp) x (20 duplications)]. The overall M.F. in this case would be 21.9%. Thus, despite the relatively high error rates associated with RTs, the accuracy of the PCR enzyme is expected to contribute to overall mutation frequency due to the exponential amplification of the
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