Tips from the Developer
How did you get started with the PCR method?
ANKENBAUER: I did my thesis about DNA polymerases in 1982, just one year before Kary Mullis invented the technique. So in the nineties, after Taq polymerase had been established, my job at Boehringer Mannheim (now Roche) was to find polymerases with additional features. It was about looking for high fidelity enzymes or polymerases for reverse transcription PCR.
Do you remember your first PCR?
ANKENBAUER: Indeed I do. My very first experience with the PCR method was a rather difficult one. We used forward primers to fish for homologous regions in polymerase fragments from thermophiles. For that purpose, we designed a set of primers but we didn't know how good they would bind. It was a bit challenging. But in the end we were rather successful. In contrast to other teams, we were fortunate to fish an enzyme from the archaeon Thermococcus gorgonarius, which had no introns and was relatively short. In 1997, we filed a European patent for that Tgo polymerase, which is 25-fold more accurate than Taq DNA polymerase due to its 3'-5' proofreading exonuclease activity.
What has changed from the early days of PCR until today?
ANKENBAUER: In its beginnings, PCR was a highly sophisticated matter. At that time, researchers had to be aware of what they were required to pipet for each of their individual applications. However, with the entry of master mixes, PCR became more robust and more convenient. Today's master mixes are designed for generic use. That means you can amplify up to 85% of all PCR targets without requiring any further empirical optimization except temperature adaption. Moreover, in the nineties, new PCR polymerases and enzyme blends entered the market allowing for a lot of new applications. We were the first to introduce enzymes for RT-PCR and GC-rich PCR. Further progress came from the development of hot start polymerases, which are not active below the elongation temperature and thus prevent non-specific amplification (e.g., of primer dimers) during the initial stages of the PCR. Finally, PCR has become much faster because thermocyclers with high ramp rates have been developed as well as buffer formulations allowing optimized amplification.
What remains unchanged?
ANKENBAUER: The success rate of primer design is the same as it ever was. But primer design has become significantly cheaper and faster, and today's design algorithms can indicate alternative hybridization sites. Likewise, the specificity of the amplification remains the major challenge of every PCR. Potential side reactions in the first PCR cycles, such as elongation of primer dimers, compete with the amplification of the initially low-abundance target sequences. However, specificity has been significantly improved since the introduction of hot start polymerases.
What is necessary to have a stable and reliable PCR?
ANKENBAUER: You need a stable and active polymerase, and you have to find the optimal nucleotide concentration to have a rapid and specific amplification. The higher the molarity of dNTPs, the faster the reaction. Another important factor is the concentration of the magnesium ions that act as cofactors for the polymerase. If it's too high, the reaction can become unspecific because the polymerase jumps from strand to strand and only adds a few nucleotides to the primers resulting in low processivity. If you want to amplify a target specifically and uninterrupted, you have to reduce the amount of dNTPs and Mg2+. Another crucial factor is the pH and the salt concentration. The higher the pH, the easier the template DNA dissociates during heating, and the less likely it is that unspecific primer binding occurs. A high salt concentration helps the polymerase to remain fixed to the template and stabilizes primer binding. This can be further supported by supplements such as Betain or DMSO.
What are key winning factors?
ANKENBAUER: If you have found the right primer and the appropriate master mix, you will have a high success rate. If you still have different PCR products, you have to raise the annealing temperature stepwise. Most often, the chosen primer is too long, resulting in primer dimerization. Traces of DNA in RNA samples or pseudogenes are also factors that can ruin a PCR result. You have to select a primer that prevents amplification of these non-target molecules.
DNA contamination can also ruin a PCR result. Is there an easy strategy to prevent it?
ANKENBAUER: Carryover of products from a previous PCR experiment is a serious threat to every PCR experiment. Even minute amounts can lead to false positive results. If you are using Taq polymerase, an easy way to prevent carryover contamination is to incorporate dUTPs instead of dTTPs in every PCR. Before you start a new PCR, pretreat your PCR mix with Uracil-DNA N-glycosylase (UNG), which eliminates all deoxyuracil-containing amplicons during the initial denaturation step. After treatment, only fresh template remains for amplification. However, that procedure doesn't work for B-type polymerases such as Pwo polymerase, because UNG blocks enzymes with an exonuclease activity. In these cases, physical separation of the areas for DNA extraction, PCR setup, and amplification is the best choice. This also helps to prevent cross-contamination of your samples.
What is the future of PCR?
ANKENBAUER: Robust polymerases, which are resistant to PCR inhibitors, can further improve PCR, allowing for amplification of PCR targets from body fluids (such as blood) or raw cell lysates.