Polymerase chain reaction (PCR) is an established method to amplify specific fragments of DNA, for applications such as gene cloning, genetic fingerprinting and diagnosis of disease. Although PCR reactions have historically been set up at room temperature, this approach is highly susceptible to non-specific amplification. Such non-specific amplification often complicates data analysis and can even lead to a failed experiment. To address the problem of non-specific amplification, Hot Start PCR methods have been evolved which inhibits the activity of Taq DNA polymerase at room temperature (Figure 1). Hot start PCR offers many advantages such as:
- Minimize non-specific amplification
- Convenient room temperature reaction setup
- Prevents mispriming and primer dimer formation
- Appropriate for specific annealing of the primers and their extension
- High yield and specificity of target amplicons
- Wide range of amplicon length (up to 10kb)
- Suitable for high throughput applications including automated liquid handling platforms
- Enhance data reproducibility
Figure 1. A. Principle of aptamer based hot start PCR. B. Formation of by-product and associated risk of false positive detection is greatly reduced by Hot Start Pol. C. Hot Start DNA Polymerase amplifies from plasmid template amounts as low as 1 femtogram (fg).
Hot Start PCR predominately includes three distinct methods:
- Antibody-mediated where one or several Taq specific antibodies bound at the enzyme’s active site denatures in the heat activation step allowing Taq polymerase to function.
- Aptamer-mediated where Taq polymerase function due to aptamer dissociating from the enzyme at a lower temperature and shorten protocols by eliminating the need for a specific heat activation step (Figure 1).
- Or the incorporation of specialized Hot Start dNTPs into protocols (modified dNTPs with thermolabile protecting group dictate when the reaction will begin).
An advantage of aptamer-mediated approach is that aptamers dissociate from the enzyme at a lower temperature than heat-labile blocking groups or antibodies, accelerating PCR protocols by eliminating the need for a high temperature activation step. Moreover, aptamer-based inhibition is fully reversible, meaning that Taq DNA polymerase activity is blocked at the end of thermal cycling.
Based on this experience and taking advantage of aptamers, Rutschke et al., developed a hot start concept for the reverse transcriptase (RT) to improve the performance of real time RT-PCR assays. This way, the authors demonstrated that a hot start RT can be generated by using an aptamer directed against moloney murine leukemia virus (M-MLV) RT for the detection of Middle East respiratory syndrome coronavirus (MERS-CoV). Results with aptamer revealed a reduced RT activity at low temperatures which achieving full activity at the specific annealing temperature of 55°C. Sensitivity (limit of detection 95%) of the MERS CoV assay was increased by two times in the presence of aptamer compared with standard RT (Figure 2).
Figure 2. Real-time RT-PCR was carried out to determine concentration-dependent hit rates (A), which were analyzed in a probit regression (B). Sensitivity of the MERS-CoV assay was increased by about two times in the presence of aptamer.
In summary, the authors could demonstrate that hot start RT has the potential to improve the sensitivity of real-time RT-PCR assays. As PCR is an integral part of the aptamer selection process, Aptamer Group LTD uses highly optimized hot start PCR reagents to deliver the highest quality aptamers. To know more about our selection process and help with your research, please complete the form below.
- https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma Aldrich/General_Information/2/hot-start-pcr-ebook-mk.pdf
- Rutschke N., Zimmermann J., Moller R., et al. (2015). Hot start reverse transcriptase: an approach for improved real-time RT-PCR performance. Journal of Analytical Science and Technology. 6:20. DOI 10.1186/s40543-015-0063-4