Curr. Issues Mol. Biol. 16: 1-6. OnlineFuture journal Trends at http://www.cimb.org in RT-PCR 1 New Developments in Quantitative Real-time Polymerase Chain Reaction Technology Vijay J. Gadkar and Martin Filion* Isothermal detection methods The current RT-qPCR technology is based on the classical Department of Biology, Université de Moncton, three-step thermal cycling process which is, template 18 Antonine-Maillet, Moncton, NB E1A 3E9 Canada denaturation, followed by primer/probe annealing and fnally, extension/detection of the fuorescence signal, to Abstract amplify and detect the target transcripts all under real-time Real time-quantitative PCR (RT-qPCR) technology has conditions (Gelfand, 1992). A very commonly observed revolutionized the detection landscape in every area phenomenon in this multistep thermo-cycling amplifcation of molecular biology. The fundamental basis of this system is the generation of spurious fuorescence signal technology has remained unchanged since its inception, due to mispriming of primer/probes. To overcome such however various modifcations have enhanced the overall limitations, detection platforms have been proposed performance of this highly versatile technology. These which amplify the target exponentially like PCR, but improvements have ranged from changes in the individual under isothermal conditions, i.e. at a fxed, user-defned components of the enzymatic reaction cocktail (polymerizing temperature (reviewed by Gill and Ghaemi, 2008). enzymes, reaction buffers, probes, etc.) to the detection The helicase-dependent (HDA) amplifcation system is system itself (instrumentation, software, etc.). The RT- one such novel ‘non-PCR’ system for amplifying target DNA qPCR technology currently available to researchers is more (Vincent et al., 2004) and RNA (Goldmeyer et al., 2007), sensitive, faster and affordable than when this technology under isothermal conditions. This revolutionary amplifcation was frst introduced. In this article, we summarize the system makes use of a novel enzymatic cocktail which developments of the last few years in RT-qPCR technology does not require the DNA to be cycled between different and nucleic acid amplifcation. temperatures, like that done for reactions based on Taq DNA polymerase amplifcation or any of its variants. In lieu Introduction of a standard denaturation step, the HDA system uses the Since its introduction in the late 1990s, real time-quantitative helicase enzyme to unwind the double-stranded DNA and PCR (RT-qPCR) technology has revolutionized the detection with the aid of other polymerizing enzymes, an exponential landscape in every aspect of molecular biology (Filion, 2012). amplifcation is achieved (Vincent et al., 2004). All these The fundamental basis of this technology has remained steps are performed at a fxed, user defned temperature. unchanged since its inception, which is fuorescence based Though extremely novel when frst introduced, the HDA detection of its target, amplifed via classical multistep PCR. system suffers from one major limitation- its inability to A steady modifcation(s) of individual components which amplify DNA targets greater than 200 bp (Vincent et al., makeup the RT-qPCR platform has however enhanced 2004). As a result, in its present state, it is seriously unable the overall performance of this highly versatile technology. to challenge and act as a viable alternative to the highly These improvements have ranged from changes in the versatile PCR, or any amplifcation system based on it. individual components of the enzymatic reaction cocktail Despite this technical limitation, HDA-based (polymerizing enzymes, reaction buffers, probes, etc.) to amplifcation is increasingly being viewed as an attractive the detection system itself (instrumentation, software, etc.). alternative in any amplifcation system, where length of the As a result, the RT-qPCR technology currently available to amplicon is less than 200 bp. One excellent example of researchers is more sensitive, faster and most importantly, such short amplicon size detection system is RT-qPCR. It affordable for laboratories on moderate/modest budgets, is a well known fact that, for an effcient amplifcation, the than when this technology was frst introduced (Filion, primer/probe parameters of the RT-qPCR detection system 2012). is usually within the 50–150 bp size range. It is therefore not In this article, we seek to briefy summarize some of impossible to develop a RT-qHDA detection system, where the developments which have happen within the last few the classical Taq DNA polymerase amplifcation cocktail years in the area of RT-qPCR technology and nucleic acid is substituted by an enzymatic system, which amplifes its amplifcation (e.g. classical PCR). In our opinion, these target at a fxed (isothermal) temperature. developments might have a direct relevance for researchers Recently, Tong et al. (2008) showed that such working in the area of environmental transcriptomics. Some enzymatic substitution is indeed feasible. In their work, of described examples in this article have been validated primer/probes designed for TaqMan RT-qPCR fuorescence on RT-qPCR platforms, making it relatively easy to adapt in system were used to develop a novel RT-qHDA system to their current state, while others have only been implemented detect various biothreat microorganisms. Since pre-existing on a conventional end-point detection platform. TaqMan primer/probes were used, the RT-qHDA system was optimized to work with the 60–65°C temperature *Corresponding author: Email: [email protected] range and detection sensitivity, similar to what one would Horizon Scientifc Press. http://www.horizonpress.com 2 Gadkar and Filion expect from a classical RT-qPCR system. Though very the implementation of a highly versatile RNA based IAC, preliminary, it is tempting to conclude that the RT-qHDA called the myIC system. The myIC is essentially a 200 bp system, at least in theory, is ‘backward compatible’ with artifcial sequence, with no known homology to any nucleic a pre-existing RT-qPCR system. In other words, the user acid accession in the GENBANK (NCBI) database. As a does not have to separately invest in a new detection result, primer/probes designed for this sequence are not platform (i.e. instrumentation) or custom synthesize the expected to cross-react with any non-target nucleic acids, furogenic probes, both of which could be prohibitively typically expected to be present in high amounts in RNA/ expensive. The only modifcation one would have to make DNA extracted from complex environmental matrices. is to substitute the Taq DNA polymerase based reaction Recently, its applicability was successfully demonstrated components, with a HDA amplifcation cocktail, which is in quantifying bacterial gene transcripts from natural soil now commercially available in various formats from Biohelix samples spiked with a defned amount of the bacterium Corp, USA. Interestingly enough, isothermal amplifcation Pseudomonas sp. LBUM300 (DeCoste et al., 2011). This systems themselves are also constantly being refned to success should spur researchers to implement the myIC enhance fdelity (amplicon length) and robustness of the system to quantify microbial gene expression in other amplifcation process. Protocols which make use of novel environmental matrices, but were unable due to lack of a enzymatic cocktails which are more effcient in unwinding suitable IAC. The added advantage of its non-proprietary and amplifying the target double helix are constantly being usage and its applicability in both DNA/RNA formats reported (Kiesling et al., 2007; Tan et al., 2008; Schaerli et (Gonzalez-Escalona et al., 2009; Deer et al., 2010), makes al., 2010; Joneja and Huang, 2011). myIC a very attractive IAC to implement in various RT- In our opinion, adapting such ‘non-PCR’ amplifcation qPCR based detection systems. technologies could in the near future lead to detection platforms (Mahalanabis et al., 2010) which are more LNA/Zip nucleic acids robust and would not suffer from the inherent drawbacks, One critical factor for any PCR based detection platform, for example spurious amplifcation, cycling parameter including RT-qPCR, is the invariant ability of the primer/ standardization, typically associated with the classical probe to discern its target from a heterogeneous mixture three-stage PCR system. and bind it with extreme specifcity. In RT-qPCR, the design of these primers/probes is governed by strict guidelines Development of novel normalization controls (hybridization/sequence parameters) which, in turn, are The frst step towards analysing microbial gene expression based on the fuorescence chemistry used for that particular requires a quantitative extraction of RNA. This step has assay. For example, the hybridization parameters for proven to be highly problematic for environmental matrices, TaqMan assays are different from the Molecular beacon due to compounded ineffciencies in individual steps based furogenic assays. Empirically, these primers/probes which include, but not limited to, incomplete cell lysis, are designed with the help of specifc software’s (e.g. RNA degradation by ubiquitous RNases, co-extraction PrimerExpress™ for TaqMan detection system) which are of inhibitors and their interaction with the enzymes used proprietary and usually supplied with the detection platform. (Lorenz and Wackernagel, 1987; Moran et al., 1993, Alm Since these software use specifc built-in algorithms, with and Stahl, 2000). One straightforward