Maximizing PCR and RT-PCR Success

PCR Brochure 10/2016 5 Reverse transcriptases RT-PCR allows the analysis of RNA using a combination of reverse transcription and PCR. cDNA is synthesized from RNA templates using reverse transcriptases — RNA- dependent DNA polymerases normally isolated from a variety of retroviral sources (e.g., from Avian Myeloblastosis Virus [AMV] or Moloney murine leukemia virus [MMLV]). Although thermostable DNA polymerases such as Tth DNA polymerase also exhibit reverse transcriptase activity under specific conditions, these enzymes are not as efficient for reverse transcription as mesophilic reverse transcriptases. The single-stranded cDNA produced by reverse transcription is more susceptible to nonspecific primer annealing at lower temperatures than double-stranded DNA (e.g., genomic DNA). Nonspecific annealing can result in poor amplification specificity which, especially when combined with limiting cDNA quantity or low transcript abundance, leads to reduced sensitivity and poor reproducibility. Amplification specificity is crucial for successful RT-PCR and is best achieved by combining innovative buffer solutions with specially modified reverse transcriptases and hot-start PCR. The use of optimized reverse transcription buffers and specially developed reverse transcriptases (such as Omniscript® and Sensiscript® contained in the QIAGEN OneStep RT-PCR Kit and QIAGEN OneStep Ahead RT-PCR Kit) can resolve secondary structures that commonly occur with single-stranded RNA molecules. To further increase specificity and enable room-temperature setup, the QIAGEN OneStep Ahead RT-PCR Kit includes an RT-blocker that keeps the reverse transcriptase inactive at ambient temperatures; therefore preventing it from nonspecific amplification of primer–dimers. When the reaction is heated to the catalytic optimum of 50–55°C, the blocker dissociates from the RT enzyme rendering it fully active (Figure 5). Figure 4. Highly reliable and sensitive PCR with QIAGEN‘s HotStar HiFidelity DNA Polymerase Kit. PCR was performed using the HotStar HiFidelity DNA Polymerase Kit and a high-fidelity DNA polymerase from Supplier F and analyzed by A agarose gel electrophoresis and by B the QIAxcel® Advanced System. Amplicons of 1.5 kb (lanes 1 and 3) and 750 bp (lanes 2 and 4) were generated using 100 ng human genomic DNA as a template. HotStar HiFidelity DNA Polymerase provided higher yields and more sensitive results compared with the polymerase from Supplier F. Supplier F 4 1 M QIAGEN 2 3 Supplier F 4 1 M QIAGEN 2 3 A B 23 23

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