from Lia C.R.S. Teixeira and Etienne Yergeau writing in Quantitative Real-time PCR in Applied Microbiology:

Quantitative polymerase chain reaction (qPCR) represents an effective method to quantify genes or transcripts within environmental samples. For that reason, qPCR has been widely used to characterize the functional patterns of complex microbial communities. In this chapter we summarize some recent applications of different qPCR approaches targeting functional genes encoding key enzymes in the N-, C- and S-cycles and also functional genes related to antibiotic resistance. We also point out some limitation of qPCR approaches. The ongoing development of new molecular techniques, like metagenomics, will have positive impacts on the specificity and the coverage of qPCR assays, since the availability of more sequence data will help to improve the design of primers targeting functional genes.

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

from Claudia Goyer and Catherine E. Dandie writing in Quantitative Real-time PCR in Applied Microbiology:

Development of quantitative PCR (qPCR) has facilitated major advances in assessment of microbial community abundances in complex environmental samples including water, soil, sediments, compost and manure and in our understanding of factors influencing community sizes in situ. qPCR has increasingly been used in environmental studies due to its sensitivity, ease of use, and the capacity to run large numbers of samples. However, qPCR has some limitations, which are specifically caused by the nature of environmental samples, including the variability in microorganism distribution, the efficiency of DNA recovery and purification, and the amount and type of PCR inhibitors co-extracted with the target nucleic acids. The heterogeneity of the templates amplified by qPCR can generate PCR biases and artifacts. Accuracy of the quantification of broad groups of microorganisms is influenced by the number of gene copies per genome of the selected marker. In this review, we will discuss the main experimental considerations for using qPCR in environmental studies, including the factors affecting key steps in the process of performing quantification of microorganisms in environmental samples. Although quantification of microorganisms is challenging, it is possible to reliably quantify microorganisms in complex environmental samples using qPCR. We will also briefly review the findings of studies which have used qPCR to quantify microorganisms from complex matrices.

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

from Vijay J. Gadkar and Martin Filion writing in Quantitative Real-time PCR in Applied Microbiology:

Environmental matrices are highly diverse in their composition and range from simple (e.g. water) to highly complex (e.g. organic soils/biosolids). Analysis of microbial gene expression from such substrates is done for variety of purposes which could range from bio-surveillance to elucidation of biological function of a target microbe. Quantitative real-time PCR (RT-qPCR) has become a technique of choice for studying such bio-processes, due to its unique ability to both detect and quantify a target transcript in real-time. Challenges in extracting inhibitor-free, structurally intact RNA, amenable for a sensitive technique like RT-qPCR, has however proved to be a major impediment in our ability to rigorously implement this highly versatile technology. Despite these "substrate defined" limitations, many attempts have been made to implement the RT-qPCR technology. Efforts like these have given us invaluable insight into the expression status of a particular transcript and hence, the biological functioning of the microbe, specifically under natural in situ conditions. As a result, it has enhanced our understanding of the role and diversity of many microbial populations which, previously was not possible using conventional molecular approaches. In this chapter, we have sought to summarize such technical problems faced by molecular environmental microbiologist and solutions developed to mitigate those challenges.

Further reading: Quantitative Real-time PCR in Applied Microbiology

from Luca Cocolin and Kalliopi Rantsiou writing in Quantitative Real-time PCR in Applied Microbiology:

Since its first application in food microbiology in the late '90s, quantitative PCR (qPCR) has attracted the interest of researchers, working mainly in the field of food safety, but lately also of microbiologists studying spoilage and fermentation processes. In addition to the advantages that conventional PCR offers in microbiological testing, i.e. specificity, reduced time of analysis and detection of viable but not culturable cells, qPCR allows quantification of target populations. This aspect is particularly relevant for foodborne pathogens, for which specific microbiological criteria exist, but also for spoilage and technological important microorganisms, in order to follow their population kinetics in foods. Although advancements in food microbiology have been made from its application, qPCR has not yet been utilized to its full potential: the quantification step is only rarely carried out and qPCR is often used as an alternative of conventional PCR. In this chapter we will critically describe the application of qPCR in food microbiology based on the available literature, taking into account the specific problems and suggesting some possible solutions.

Further reading: Quantitative Real-time PCR in Applied Microbiology

from Mathilde H. Josefsen, Charlotta Löfström, Trine Hansen, Eyjólfur Reynisson and Jeffrey Hoorfar writing in Quantitative Real-time PCR in Applied Microbiology:

The polymerase chain reaction has revolutionized the world of scientific research and its broad application has caused a tremendous development of versatile PCR instruments and chemistries to fit its purpose. This chapter provides the reader with a general introduction to the basics of real-time PCR instrumentation, including the thermal and optical systems and the software. Performance parameters such as temperature uniformity, accuracy and ramp speed as well as reaction format, optical systems, calibration of dyes, software and comparison between different real-time PCR platforms will be discussed from a user perspective leading to an instrument selection guide. Differences between fluorescent DNA binding dyes and target-specific fluorescently labeled primers or probes for detection of amplicon accumulation will be discussed, along with the properties and applications of the most frequently applied chemistries. The fluorophores and quenchers used for primer and probe labeling and their compatibility will be presented, and finally the future challenges and trends within the field of qPCR instrumentation will be discussed.

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

from Mikael Kubista, Vendula Rusnakova, David Svec, Björn Sjögreen and Ales Tichopad writing in Quantitative Real-time PCR in Applied Microbiology:

As the qPCR field advances, the design of experiments and the analysis of data is becoming more important and more challenging. Calculation of relative expression of a reporter gene to a reference gene in pairs of samples using the ΔΔCq method is no longer sufficient. Studies are now designed using multiple markers, nested levels, exploring or confirming the effect of multiple factors, occasionally in paired designs, etc. Proper handling of such data requires software that support the planning and design of experiments, and data analysis. Several software with these capacities are emerging. This chapter describes some of the features of one of the most powerful of those: GenEx from MultiD Analysis.

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

from Vijay J. Gadkar and Martin Filion writing in Quantitative Real-time PCR in Applied Microbiology:

Real time-quantitative PCR (RT-qPCR) technology has revolutionized the detection landscape in every area of molecular biology. The fundamental basis of this technology has remained unchanged since its inception, however various modifications have enhanced the overall performance of this highly versatile technology. These improvements have ranged from changes in the individual components of the enzymatic reaction cocktail (polymerizing enzymes, reaction buffers, probes, etc.) to the detection system itself (instrumentation, software, etc). The RT-qPCR technology currently available to researchers is more sensitive, faster and affordable than when this technology was first introduced. In this chapter, we summarize the developments of the last few years in RT-qPCR technology and nucleic acid amplification.

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

from Stephen A Bustin, Sara Zaccara and Tania Nolan writing in Quantitative Real-time PCR in Applied Microbiology:

The real-time fluorescence-based quantitative polymerase chain reaction (qPCR) has become the benchmark technology for the detection of nucleic acids in every area of microbiology, biomedical research, biotechnology and in forensic applications. Unlike conventional (legacy) PCR, which is a qualitative end-point assay, qPCR allows accurate quantification of amplified DNA in real time during the exponential phase of the reaction. The cost of instruments and reagents is well within reach of individual laboratories, assays are easy to perform, capable of high throughput and combine high sensitivity with reliable specificity. It is possible to achieve accurate and biologically meaningful quantification if meticulous attention is paid to the details of every step of the qPCR assay, starting with sample selection, acquisition and handling through assay design, validation and optimisation. The growing awareness of the need for standardisation, quality control and the significant problems associated with inadequate reporting of the assay has resulted in the publication of guidelines for minimum information for the publication of qPCR experiments (MIQE).

Further reading: Quantitative Real-time PCR in Applied Microbiology   Related publications

Theodoros N. Rampias, Emmanuel G. Fragoulis and Diamantis C. Sideris

Cloning of alternatively polyadenylated transcripts is crucial for studying gene expression and function. Recent transcriptome analysis has mainly focused on large EST clone collections. However, EST sequencing techniques in many cases are incapable of isolating rare transcripts or address transcript variability. In most cases, 3 ́ RACE is applied for the experimental identification of alternatively polyadenylated transcripts. However, its application may result in nonspecific amplification and false positive products due to the usage of a single gene specific primer. Additionally, internal poly(A) stretches primed by oligo(dT) primer in mRNAs with AU-rich 3 ́UTR may generate truncated cDNAs. To overcome these limitations, we have developed a simple and rapid approach combining SMART technology for the construction of a full length cDNA library and hybrid capture PCR for the selection and amplification of target cDNAs. Our strategy is characterized by enhanced specificity compared to other conventional RT-PCR and 3 ́ RACE procedures.
Recommended reading

from Gregory L. Shipley writing in PCR Troubleshooting and Optimization: The Essential Guide:

The MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines have been presented to serve as a practical guide for authors when publishing experimental data based on real-time qPCR. Each item is presented in tabular form as a checklist within the MIQE manuscript. However, this format has left little room for explanation of precisely what is expected from the items listed and no information on how one might go about assimilating the information requested. This chapter presents an expanded explanation of the guideline items with commentary on how those requirements might be met prior to publication.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Ian Kavanagh, Gerwyn Jones and Saima Naveed Nayab writing in PCR Troubleshooting and Optimization: The Essential Guide:

Whilst qPCR is a powerful technique, the results achieved using this method is valid only if the appropriate controls have been included in the experiment. Careful selection of controls and proper optimisation of qPCR conditions promise generation of highly specific, repeatable, reproducible and sensitive data. This chapter discusses the strategies for preparing both negative and positive controls for PCR, when they should be employed and how to interpret the information they provide. It also highlights the significance of standard curves for determining the initial starting amount of the target template and for assessing assay efficiency, precision, sensitivity, and dynamic range. It also provides guidance on how to prepare standards, interpret standard curve and troubleshoot inefficient qPCR reactions.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Martina Reiter and Michael W. Pfaffl writing in PCR Troubleshooting and Optimization: The Essential Guide:

PCR technology is based on a simple principle; an enzymatic reaction that increases the amount of nucleic acids initially present in a sample but this powerful method makes it possible to detect specific mRNA transcripts in any biological sample by the application of RT-PCR. The RT-PCR quantitative analysis workflow has several steps, each of which is crucial to the success of the experiment. It starts with a sampling step, followed by nucleic acid extraction and stabilization, cDNA synthesis and finally the qPCR where the mRNA quantification takes place. PCR itself is quite a stable reaction with reproducibility between 2-8% but the number and nature of the pre-PCR steps mean that there are many sources of experimental variance in the workflow. Reliable data can only be produced when the experimental variance is minimized, so the sources of variation must be identified and optimized for each step of each experiment. Typically, however, the pre-PCR steps are neglected and optimization is done for PCR reaction only. In this chapter the optimization of the whole RT-PCR workflow will be discussed and recommendations to reduce experimental variance and produce more reproducible and reliable results are put forward.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Sandrine Javorski-Miller and Ivan Delgado Orlic writing in PCR Troubleshooting and Optimization: The Essential Guide:

A paper from 2008 mentions that quantitative PCR is 25 years old (VanGuilder et al., 2008) but routine use of this technology has only taken off during the past 12 years. The first commercial Real-Time PCR instrument, the ABI Prism 7700, was introduced to researchers in 1996 by Applied Biosystems (Gibson et al., 1996; Heid et al., 1996). Since then over 40 additional Real-Time PCR instruments have been developed by more than a dozen vendors. Because there are so many Real-Time PCR instrument available utilizing a wide range of technologies, scientists face a daunting selection task. The space includes everything from entry level (single color detection, a small number of samples, low cost) to more complex (over 5 channel colors and multiplex detection, thousands of samples processed in each run, and expensive system price). In this chapter we highlight some key features that differentiate Real-Time PCR instruments, with the goal of simplifying the criteria needed to select the instrument that best fit a specific scientist's research needs.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Jan Hellemans and Jo Vandesompele writing in PCR Troubleshooting and Optimization: The Essential Guide:

Real-time quantitative PCR (qPCR) is the gold standard for fast, accurate, sensitive and cost-efficient gene expression analysis. Despite its conceptual simplicity and ease of use, the multi-step qPCR workflow contains many potential pitfalls. An intelligent experiment design and setup, high quality reagents and assays, quality controls in each step of the workflow, proper quantification models and appropriate bio-statistical analyses pave the way to successful gene expression results. This chapter will cover all data analysis aspects from the evaluation of pilot studies and quality controls, through universally applicable quantification models and bio-statistics, to the reporting of experiment results.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Gavin Meredith, Miro Dudas, Mark Landers, Vasiliki Anest, Jonathan Wang, Caifu Chen, Peter Jozsi and Christopher Adams writing in PCR Troubleshooting and Optimization: The Essential Guide:

The field of epigenetics transcends traditional genetics, genomics, molecular biology, and is poised to revolutionize the field of medical research and healthcare. It is a diverse field that encompasses the study of nuclear components such as chromatin structure, including histone modifications, protein/DNA interactions, protein/RNA interactions, and how these factors influence gene function. It also includes the study of DNA methylation and the role that non-coding RNAs play in influencing DNA methylation patterns, chromatin structure and ultimately regulating gene expression. Just as the field of epigenetics is broad and complex, so is the molecular technology of polymerase chain reaction (PCR). For every question one would like to address in any of these areas of epigenetics, there is a PCR application and instrumentation suitable to address it. For example there are numerous PCR-based approaches to look at DNA methylation patterns, densities, and even the methylation status of individual cytosine residues by PCR. Additionally, there are PCR methods to survey ncRNA expression and identify regions of the genome where proteins and RNA interact or where certain functional histone marks are located. This chapter provides an overview of these methodologies with a focus on the advantages and disadvantages of each approach.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Cameron N. Gundry and Matthew D. Poulson writing in PCR Troubleshooting and Optimization: The Essential Guide:

PCR is a highly sensitive and specific technique used in molecular biology laboratories everywhere. It is able to provide near 100% sensitivity and specificity with appropriately designed assays in controlled situations. However, results do not always match this potential. The most common problems in PCR arise from overlooking basic principles in assay design and optimization. Maximum PCR performance depends on key factors which include: 1) choosing an appropriate detection system, 2) using available software for the best primer and probe design, 3) assessing sample quality and controlling inhibitors, 4) avoiding amplicon and environmental contamination, 5) optimizing for reagent quality and concentration, and 6) modifying the thermal cycling protocol for optimal sensitivity and specificity. This chapter will address all of these factors to aid the investigator in designing high quality PCR assays.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from N. Reginald Beer and John H. Leamon writing in PCR Troubleshooting and Optimization: The Essential Guide:

PCR has traditionally been performed in microliter-scale reactions because larger scale volumes are prohibitively expensive and wasteful while the smaller scales (nanoliter and below) are impractical with available sample handling tools and detection systems. At the microliter scale, samples can contain mutually competitive and distinct targets, introducing amplification bias and competitive inhibition that degrade assay performance. Microfluidic Emulsion PCR has emerged as a technique to resolve these challenges by a combination of two enabling technologies. Emulsion PCR provides the advantages of fluid partitioning, namely elimination of sample bias and the ability to run millions of reactions in discrete volumes, while microfluidics simultaneously reduces the sample volume, introduces a level of control over emulsion parameters, and provides optical observability of the partitioned microreactors. Furthermore, since microfluidic emulsions can be made monodisperse in size, they allow the assumption of an average dilution per reactor to permit the exploitation of Poisson statistics for very accurate titer estimation. Microfluidic emulsions can also be employed to perform solid-phase amplification with bead-based assays, combining yet another useful technique with the sample partitioning benefits of droplets. We expect the advantages of both emulsion PCR and microfluidics will encourage new applications and the integration of these enabling technologies will improve PCR performance.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Carl T. Wittwer and Jared S. Farrar writing in PCR Troubleshooting and Optimization: The Essential Guide:

The polymerase chain reaction (PCR) has become a fundamental tool in molecular research and clinical testing. The origins of PCR and its early evolution are described, including adaptation to RNA, thermostable polymerases, automation, improvements in specificity and rapid temperature cycling. Perhaps the most significant advance is real-time PCR, combining both amplification and detection into one instrument as a superior solution for nucleic acid quantification. Real-time PCR is enabled by monitoring the reaction with double stranded DNA dyes or specific probes, including hydrolysis, hybridization, and conformation-sensitive probes. Early real-time instruments are compared. PCR product and probe melting analysis continues to improve in resolution, allowing greater sequence detail for genotyping and variant scanning. Microfluidic platforms and digital PCR are destined to find more applications in the future.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from John F. Mackay and Carl T. Wittwer writing in PCR Troubleshooting and Optimization: The Essential Guide:

Real-time qPCR using SYBR Green and melting curve analysis to verify specific product amplification has become a standard laboratory technique for rapid, high throughput gene quantification. An extension of this melting curve method - High Resolution melting analysis (HRMA)_ is now doing the same for the analysis of sequence variation, allowing rapid cost-effective discrimination of sequences to SNP level in an automated closed-tube method. Two PCR primers are typically required as with SYBR Green quantification but HRMA differs in its requirement for the use of a saturating dye, precise reaction temperature control and software algorithms to cluster the melting curves. Originally described for SNP analysis (and still the leading application), HRMA is now being used in a wider context- HLA comparisons, microsatellite genotyping and methylation status of DNA sequences. New developments such as unlabeled probes and snapback elements on the PCR primers allow the simultaneous genotyping of a desired SNP with the scanning of the whole amplicon for other sequence variation. This chapter covers some of these developments and provides a guide to those wishing to establish this technique, as well as troubleshooting advice for those already underway.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

from Jack M. Gallup writing in PCR Troubleshooting and Optimization: The Essential Guide:

One of the least-acknowledged problems with PCR, RT-PCR and qPCR is reaction inhibition. Addressing or eliminating inhibition is central to allowing qPCR to be modeled by the least complex mathematics, and enables more effective troubleshooting of amplifications from difficult templates such as AT- or GC-rich sequences, repetitive sequences, and templates with prohibitive secondary structures. In the absence of inhibition, additives aimed at improving PCR, RT-PCR and qPCR performance can be assessed more directly, allowing investigators to identify and utilize better primer/probe designs, enzymes and master mixes, and formulate better reverse transcription reactions. In addition to inhibition, RNA integrity is another major concern which must be addressed both by using appropriate optical assessments and the 3':5' assay. To address inhibition, commercial kits for removing inhibitory substances have been developed in addition to the SPUD assay and the P-Q assay-development/project-management software tool. Although reagent choice alone plays a large part in determining the success or failure of reverse transcription, PCR, RT-PCR or qPCR, this chapter briefly explores some of the current strategies for detecting, avoiding and/or eliminating inhibition during reverse transcription, PCR, RT-PCR and qPCR. It also discusses strategies to amplify difficult templates and optimize reverse transcription reactions.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide

With 3 billion bases in the human genome, it is not easy to find and analyze the small sequence regions that confirm a genetic disorder, identify an oncogenic change or detect microbial infection. The polymerase chain reaction (PCR) provides this focus. Since its development 25 years ago, it has become the most important tool for working with nucleic acids in molecular biology and clinical diagnostics. It deserves such central recognition because of its simplicity.

Before PCR, molecular methods were multi-stepped, laborious and time consuming. To amplify DNA, it had to be cloned into plasmids, the plasmids inserted into bacteria, the bacteria grown in culture, the bacteria harvested, the plasmids isolated from the bacteria, and the DNA inserts separated from the plasmid DNA. Southern blotting required multiple steps of restriction enzyme digestion, electrophoresis, blotting onto membranes, hybridization with radioactively-labeled oligonucleotide probes and development on X-ray film. These early techniques required large amounts of DNA and strong technical expertise for consistent results.

PCR greatly reduced the number of steps required to generate appreciable quantities of DNA necessary for many applications. The acceptance of PCR in the scientific community was relatively swift, with an independent research group using the technique within a year. PCR has revolutionized molecular biology and clinical diagnostics. (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization)

PCR is simple and elegant. It is remarkably robust and tolerates the addition of many diverse reagents such as electrophoresis dyes (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

PCR specificity under ideal conditions is extraordinary. However, the genomes are large and primers may bind not only to their intended target but also to other areas of the genome. Furthermore, the primers in PCR are at high concentrations, so even minor self or cross complementation may initiate primer dimers. These side reactions can create so-called "non-specific" products other than the desired product. A number of methods have been developed to prevent primer extension at low (room) temperatures where polymerase activity, although greatly reduced, is still capable of extending primers (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

The first "hot-start" techniques relied on adding an essential reaction component (such as the polymerase) only after the reaction had reached high temperatures to favor specific primer annealing. This required opening the reaction container and increased the possibility of PCR contamination. To circumvent this problem, waxes and greases were used to physically partition reagents with a barrier that would melt at high temperature, mixing the essential reagent(s) with the other reaction components (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Instead of physical separation, polymerase activity can be inhibited at room temperature. For example, monoclonal antibodies against the active site of the polymerase can inhibit the enzyme until they denature at high temperature. Alternatively, the polymerase active site can be chemically modified by heat-labile covalent modifications that break down and activate the enzyme at high temperature. Instead of inactivating the polymerase, oligonucleotide primers or dNTPs can be modified at their 3'-end with similar heat-labile linkages (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Many different reagents are now available to augment PCR specificity, but they are usually only necessary when the template copy number is low. Nevertheless, such reagents are an easy way to increase the robustness of PCR, sometimes making optimization unnecessary. If a hot start method is required, the best method depends on the circumstances. For example, an antibody-mediated hot-start is more useful in rapid PCR because chemically modified polymerases typically require 15-30 min for activation, longer than an entire rapid-cycle PCR protocol.

Real-time PCR not only automates both amplification and detection, but integrates them so that they occur concurrently. Time, temperature and fluorescence are monitored during PCR in real-time instruments. The earliest report of continuous monitoring of PCR and acquiring fluorescence at each cycle utilizing ethidium bromide, a double-stranded DNA (dsDNA) specific dye. This allowed for a truly homogenous or "closed-tube" assay in which product amplification was combined with detection. The most important application of real-time PCR is quantification of the initial template, known as quantitative PCR or qPCR (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

The first two commercial real-time PCR platforms were the ABI 7700 and the LightCycler. The LightCycler was initially developed through a small business NIH grant. The prototype, constructed at the University of Utah, integrated rapid temperature cycling with fluorescent monitoring adapted from a flow cytometer. Idaho Technology converted the prototype to a 24-sample instrument with a small footprint and simplified optics for commercial sale. In 1997, the system was licensed to Boehringer Mannheim which was subsequently acquired by Roche that same year. A 32-sample LightCycler was released by Roche in 1998 integrating rapid-cycling, SYBR Green I, dual hybridization probes and melting curve analysis (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

The ABI 7700 was a large, plate-based 96-well instrument focused on hydrolysis probes.The 7700 used a 488 nm laser and fiber optics, in contrast to the light emitting diodes and epifluorescence optics of the LightCycler. Today there are many product offerings in the arena of real-time instrumentation. Competition has driven down the costs of instruments and reagents.

Terms such as "rapid" or "fast" are relative and vague. A 1 hour PCR is fast compared to 4 hours, but slow compared to 10 min. Furthermore, faster PCR is possible if you start with a higher template concentration or use fewer cycles. It is better to define the time required for each cycle and rapid-cycle PCR has been defined as 30 cycles in 10-30 min (See: Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization) so that each cycle is 20-60 s each. The actual time of each cycle is longer than the sum of the times programmed for denaturation, annealing and extension. Indeed, during rapid PCR the temperature is usually changing. This challenges the "equilibrium paradigm" of PCR, where 3 reactions (denaturation, annealing and extension) occur at 3 temperatures over 3 time periods each cycle (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Although intuitive, the equilibrium paradigm does not fit well with physical reality. Instantaneous temperature changes do not occur and reactions occur over a range of temperatures at different rates. More accurate is a kinetic paradigm for PCR where reaction rates and the temperature are always changing. Under the equilibrium paradigm, a cycle is defined by three temperatures each held for a time period, whereas the kinetic paradigm requires transition rates and target temperatures(Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Paradigms are not right or wrong and should be judged by their usefulness. The equilibrium paradigm is simple to understand and lends itself well to the engineering mindset. The kinetic paradigm is more relevant to biochemistry, rapid PCR and melting curve analysis. Although most commercial instruments still follow equilibrium protocols, rapid protocols are a nice match for microsystems, where small volumes and rapid PCR are natural (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Rapid-cycle PCR is used in real-time instruments such as the Roche carousel LightCycler and Cepheid's SmartCycler. Other companies now promote "Fast" protocols on more conventional thermal cyclers. Few instruments based on microtiter plates and heat blocks can approach rapid-cycling speeds and rapid PCR does not require special reagents.

PCR was destined to be a quantitative technique. By both theory and practice, a well optimized PCR doubles the amount of product each cycle for many cycles. Early attempts to harness the quantifying power of PCR were limited by dependence on end-point analysis of the products generated, either by removal of an aliquot of the reaction at predetermined cycle numbers (PCR cycle titration) or serial dilution PCR (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Additional attempts were made to measure PCR products in the log phase of the reaction or include a competitive internal control in the reaction. These methods were time-consuming and labor intensive, often using agarose gels to quantify the amount of PCR product and from this determine an initial template concentration (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Real-time PCR greatly simplified quantification. By monitoring fluorescence once each cycle, fluorescence as a surrogate of PCR product amount can be plotted against cycle number. No longer is there a need to physically sample a reaction at multiple cycles or guess when PCR is exponential. By acquiring data at all cycles, exponential data can be selected in retrospect. The exponential region is identified by plotting fluorescence on a log plot and the earliest cycle "significantly above background" chosen to correlate with the initial template amount. Such quantification cycles (Cqs) are usually determined by either a fluorescence threshold or by the maximum second derivative. In either case, these fractional cycle numbers are inversely related to the log of the initial template concentration. Technical aspects of qPCR and performance guidelines have recently been published (For details see: Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Developments, particularly in the fields of genomics and biotechnology in the last few years, have resulted in a wide range of molecular tools, principally based on the detection of nucleic acid material and its amplification. They offer a novel, more sensitive and specific way of detecting microorganisms. They can also identify organisms that would not be detected with current culture techniques and can be used to track new pathogenic entities, including variants of otherwise harmless microorganisms. There are probably very few groups of microorganisms that have not been detected with these amplification techniques and several test kits have already been commercialized. Despite the success of these molecular methods, several barriers must be overcome before they can be used to routinely assess water quality and the microbiological safety of drinking water. The relationship between detection by molecular approaches and the subsequent viability or infectivity of waterborne enteric pathogens remains a concern. In addition, methods used for purifying and concentrating the target microbes and their nucleic acids from water, so that they are free of contaminants that may interfere with the analysis, need further improvement, consolidation and simplification. Further research is also needed to develop and refine the prototype protocols into collaboratively tested methods that could be used routinely and expeditiously to evaluate the microbiological safety of water.

Owing to recent advances in nanoscience and nanotechnology, various different nanomaterials and devices have been developed that show great promise for diagnostic applications. Subsequently, many different nanotechnology-based diagnostic systems have been reported in the literature and many of these have the potential to become the next generation of diagnostic tools. Moreover, microfluidic chip-based systems such as the lab-on-a-chip technology should have a significant impact on environmental microbial monitoring by permitting detection and identification of targets within minutes at the sampling site with a sensitivity level of a single cell. However, some technical and practical problems need to be solved before their full potential can be realized. These include tight control over the synthesis and functionalization of nanomaterials, as small variations can change their properties and behaviour in diagnostic methods. Also, their implementation into routine functional devices remains a challenge, and note should be taken that many of the diagnostic systems must still be taken from proof-of-concept and evaluated with environmental samples. In this regard, some of the challenges that need to be resolved, in addition to those highlighted above, include sample processing, detection of multiple agents in a single sample, and improving the sensitivity and selectivity of the assays for application to complex environmental samples.

Advances in microfluidics and microfabrication technologies have contributed greatly to the miniaturization of biological and chemical analytical systems, allowing the handling of low volume samples, as well as reductions in reagent consumption, waste generation, costs and assay time. Micro-total analysis systems (micro-TAS), sometimes called "lab-on-a-chip", are microfabricated devices capable of performing the functions of large analytical devices in small units. These devices are fabricated in glass, silicon or polymer materials, and integrate different functions and functionalities. Some sophisticated versions can perform sample introduction and handling (e.g., cell lysis, dilution and debris removal), separation (e.g., electrophoresis, chromatography) and detection, all conducted on the chip. It is believed that micro-TAS will be particularly valuable in DNA and protein analysis, genomics and proteomics, and diagnostics.

Miniaturized immunoassays have been performed successfully with microchips. These immunoaffinity microfluidic devices are considered promising platforms to achieve rapid and sensitive immunological detection of microbial cells. Zhu et al. described a simple approach in which sample trapping and concentration steps were integrated together with whole-cell immunoassay in a silicon-based lab-on-a-chip. The immunoassay was performed by injecting the sample solution, which contained C. parvum and G. lamblia, into a microchamber. Subsequently, a solution containing fluorescently-labelled target-specific antibodies was delivered and serves to simultaneously concentrate, trap and label the targeted cells at a trapping region. Following a wash step to remove unbound antibodies, the labelled parasites were detected by epifluorescence microscopy. Compared to conventional immunoassays, the total analysis time was reduced from 2-3 h to 2-5 min, and the total consumption of reagents was reduced 20-fold. In an alternative approach, Liu et al. injected microbeads coated with a primary antivirus antibody into a microfluidic device, which are subsequently trapped in front of a pillar-type filter region. A sample containing target virions was injected into the device and virions were captured on the surface of the microbeads. This was followed by injection of a labelling solution containing a secondary antivirus antibody labelled with QDs to allow detection by epifluorescence microscopy. In comparison to a standard ELISA performed on the same marine iridovirus, the minimal detectable concentration of the target virus was improved from 360 to 22 ng/ml, the detection time was shortened from 3 h to less than 30 min, and the amount of antibody consumed was reduced 14-fold.

Considerable effort has been directed to the development of chip-based systems for miniaturized and rapid PCR. The devices consist of a chip containing wells, channels, electrodes, filters, pumps, valves and heating devices designed for buffer and sample storage, PCR and target DNA detection. Remarkably, a polymeric microchip with a 1.7 microlitre chamber containing a thermocoupler was used to successfully amplify a 500-bp DNA fragment of lambda phage in 15 cycles, in a total amplification time of 240 seconds. By making use of a PCR microchip coupled with a capillary electrophoresis (CE) chip, it was more recently demonstrated that bacterial targets as low as 2-3 cells could be amplified within a 200-nl PCR chamber and the PCR-amplified target DNA was subsequently resolved by CE within 10 min. In order to improve PCR throughput and reduce the analysis time, multi-chamber PCR microfluidics on a single chip has been reported. Also, chip devices with optical windows have been fabricated that allows for measurement of fluorescence intensity during the thermocycling process, thus providing a miniaturized version of real-time PCR. In this regard, Cady et al. developed an integrated miniaturized real-time PCR detection device equipped with a microprocessor, pumps, thermocycler and light emitting diodes (LEDs)-based fluorescence excitation/detection. Monolithic DNA purification and real-time PCR enabled fast detection of L. monocytogenes cells (10⁴-10⁷) within 45 min.

In spite of their potentially powerful application in diagnostics and environmental monitoring, the 'complete' lab-on-a-chip still requires further development. The bottlenecks blocking the realization of a truly and highly integrated chip include sample preparation and product detection. Since the source of raw template samples is varied and the sample preparation methods are diverse, the miniaturization of conventional sample preparation and functionalities on a chip remains a challenge. As for on-chip detection, the product detection methods have not advanced as rapidly as other aspects of chip development. Consequently, miniaturized ultra-sensitive detectors are required if the sensitivity of the lab-on-a-chip devices is to be improved. Moreover, additional efforts have to be made towards the validation of the methods to demonstrate the reliability of micro-TAS systems.

Melting curve analysis is a powerful and practical extension of real-time PCR. While real-time PCR focuses on collecting fluorescence at a single temperature each PCR cycle, melting analysis monitors fluorescence over time as the temperature is changing. Melting analysis fits nicely into the kinetic paradigm of PCR. Duplexes melt as the temperature increases, and the hybridization of both PCR products and probes can be monitored. Similar to "old" (slow) PCR being considered an equilibrium process, "old" (dot blot) hybridizations were performed at a single temperature. Dynamic monitoring of the entire melting curve as the temperature changes defines the entire melting transition, not just a single point (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Melting curve analysis was first integrated with real-time PCR on the LightCycler. No separations or reagent additions were required and melting analysis was fast (typically 2-15 min). The dye SYBR Green I conveniently provided quantification during PCR and melting analysis after PCR. The melting temperature of a DNA duplex is determined in large part by its sequence, G/C content and length. Specific PCR products can be easily distinguished from nonspecific PCR products. In many cases melting analysis eliminates the need for post-PCR processing such as gel electrophoresis. Genotyping by melting analysis was first demonstrated with a single hybridization probe and FRET to monitor probe melting. Different single base variants produced different probe stabilities, which were revealed by melting analysis. Later, dual hybridization probes were used for genotyping and both color and temperature multiplexing exploited. The use of a single fluorescein-labeled probe instead of two probes was a further simplification. Genotyping by melting without labeled probes was first shown with allele-specific PCR and SYBR Green I. Three primers were used, one with a GC-tail to discriminate alleles by melting temperature. Genotyping without GC-tails or labeled probes became possible with the availability of saturation dyes that detect heteroduplexes. These methods are detailed later in the section on high resolution melting analysis.

In contrast to gold nanoparticles and QDs, magnetic nanoparticles have not been used in many biological applications. Nevertheless, advances in the synthesis of monodispersed magnetic nanoparticles, ranging in size from 2 to 20 nm, has provided a basis from which to explore applications of magnetic nanoparticles in diagnostics. Magnetic nanoparticles are produced from materials that can be strongly attracted by magnets or be magnetized. They can be prepared in the form of single domain or superparamagnetic (Fe₃O₄), greigite (Fe₃S₄), maghemite (gamma-Fe₂O₃), and various types of ferrites (MeO.Fe₂O₃, where Me = Ni, Co, Mg, Zn, Mn, etc.). Bound to biorecognition molecules, magnetic nanoparticles can be used to facilitate the separation, purification and concentration of different biomolecules. To do so, biorecognition molecules such as antibodies can be immobilized on the surface of magnetic nanoparticles through covalent or electrostatic interactions. After reacting these magnetic nanoparticles with sample solutions, targeted molecules can be bound by or captured on the surface of these magnetic nanoparticles. By applying a magnetic field, these nanoparticles can subsequently be concentrated and separated from the bulk solution and identified.

Biofunctional magnetic nanoparticles, in which thiolated vancomycin was attached to FePt nanoparticles, have been used to capture and detect of a wide range of bacteria at very low concentrations within 60 min. These included capturing and detection of Staphylococcus aureus at 8 cfu/ml, S. epidermidis at 10 cfu/ml, Enterococcus faecalis at 26 cfu/ml, and E. coli at 15 cfu/ml. Although the sensitivity achieved using magnetic nanoparticles is comparable to PCR-based assays, the direct capture protocol is faster than PCR when the bacterium count is low since it obviates the need for pre-enrichment of bacteria through culturing. In an alternative approach, Ho et al. reported combining biofunctional magnetic nanoparticles with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to detect pathogenic bacteria in water. Biofunctional nanoparticles were fabricated by attaching human immunoglobulin (IgG), which binds selectively to IgG-binding sites on the cell walls of pathogens, onto the surfaces of magnetite (Fe₃O₄) nanoparticles. Using this assay, both S. saprophyticus and S. aureus were detected at concentrations of 3x10⁵ cfu/ml in aqueous sample solutions. Measuring adenosine triphosphate (ATP) bioluminescence of bacteria captured onto magnetic nanoparticles is another proposed method for detecting microorganisms. E coli was detected in milk by Cheng et al. within a short period (1 h) and with a low detection limit (20 cfu/ml).

Biofunctional magnetic glyconanoparticles have also been engineered by covently binding unmodified monosaccharide d-mannose onto iron oxide nanoparticles. These particles had the ability to recognize mannose-specific receptor sites on E. coli. Magnetic nanoparticles have been developed to sequester DNA in water and capture the DNA-nanoparticles complexes by the application of high-gradient magnetic separation. Modifying magnetite clusters with poly(hexamethylene biguanide)- and polyethyleneimine resulted in strong cationic nanoparticles which enabled the binding with DNA molecules through electrostatic forces. The cationic nanoparticles can also serve as a disinfectant by binding to the negatively charged cell envelopes of bacteria. These particles were colloidally stable in fresh and ocean water for weeks at a pH <= 10.

Magnetic microparticle-antibody conjugates (Dynabeads) are commercially available and kits have been developed for the detection of Legionella species, Cryptosporidium oocysts and Giardia cysts from concentrated water samples. Dynabeads are also available for the detection of E. coli, Salmonella and Listeria species; however the samples must be grown for 6 - 8 h in a pre-enrichment broth. Streptavidin coated Dynabeads allow researchers to design their own magnetic microparticle-antibody conjugates for specialized assays (www.invitrogen.com). Biotinylated organism-specific antibodies will bind covalently onto the streptavidin coated Dynabeads. A wide range of biotin-labeled antibodies are available from companies such as Abcam (www.abcam.com).

Despite the promise shown by biofunctional magnetic nanoparticles, some challenges regarding their widespread use have yet to be overcome. In addition to requiring a robust surface chemistry to attach bioactive molecules onto magnetic nanoparticles without laborious synthetic efforts, more precise control of the numbers and orientations of the molecules on the surfaces of magnetic nanoparticles is also required.

In 1991, Holland and colleagues at the Cetus Corporation used the 5' to 3' exonuclease activity of Taq polymerase to detect amplification products post-PCR. An oligonucleotide probe complementary to the PCR product was used with a non-extendable 3'-end and a radioactively labeled 5'-end. During amplification the polymerase degraded the probe, releasing the radioactive label as smaller fragments of the probe. However, a post-PCR radiograph was required in order to visualize the degraded probe. By replacing the radioactive label with two fluorescent labels in a FRET relationship, successful allele discrimination and later real-time monitoring were achieved. These dual-labeled fluorescent probes were hydrolyzed by the 5' to 3' exonuclease activity of Taq during PCR, separating the fluorescent labels with a loss of FRET to generate fluorescence. Specificity was enhanced over dsDNA dyes because complementation to three independent oligonucleotides (two primers and one probe) was necessary for probe hydrolysis and signal generation. Hydrolysis probes (also known by the trademark TaqMan, among others) are the most commonly used probes today (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Their popularity was advanced by simplified design and a strong commercial effort to provide synthesis services. Signal generation is produced by probe hydrolysis and is irreversible and cumulative.

In contrast to hydrolysis probes, the fluorescence from hybridization probes is reversible and depends only on probe hybridization. The first hybridization probes used in real-time PCR were dual hybridization probes consisting of two oligonucleotides, one labeled at the 3'-end the other at the 5'-end. Upon hybridization to their complementary sequences and fluorescent excitation, FRET increases. Signal generation with dual hybridization probes requires annealing of four oligonucleotides (two primers and two probes), suggesting even better specificity than hydrolysis probes. Later, single hybridization probe designs were developed, including FRET between an internally labeled primer and a single-labeled probe and deoxyguanosine quenching of a single-labeled probe (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). In contrast to hydrolysis probes that are consumed during amplification, the fluorescence of hybridization probes is reversible, enabling melting analysis. The first FDA-approved genetic tests in the US (F5 and F2 single base variants) used dual hybridization probes and melting analysis for genotyping.

Real-time PCR requires monitoring the reaction during amplification. Fluorescence is a convenient method of interrogation that only requires a clear optical path for excitation and emission. Double-stranded DNA (dsDNA) dyes and fluorescently-labeled probes are both commonly used. dsDNA dyes directly measure the amount of double-stranded product produced. Probes used in real-time PCR function indirectly through fluorescence resonance energy transfer (FRET) or fluorescence quenching. Initially proposed in the late 1940s, it was not until the 1980s that FRET was applied to DNA (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). However, real-time monitoring with probes was only achieved several years later after dsDNA dyes were established in real-time PCR. One advantage of probes over dsDNA dyes is multiplexing by color with different fluorescent dyes. Nevertheless, this advantage comes at a cost in instrumentation and analysis complexity. Furthermore, multiplex analysis with dsDNA dyes is possible by melting temperature separation of products and/or probes.

dsDNA dyes are commonplace in the molecular biology laboratory. Although ethidium bromide was first used in real-time PCR, SYBR Green I is by far the most common dye in real-time PCR today. Introduced along with the LightCycler, it is more fluorescent than ethidium bromide and is easily excited at the same wavelength as fluorescein. Most real-time PCR is performed with dsDNA dyes for reasons of cost and convenience. Any PCR can be monitored with SYBR Green I. However, because dsDNA dyes are generic, there is a risk of non-specific detection of alternative PCR products. This risk can be partly eliminated by acquiring fluorescence at a temperature where only the desired product is double-stranded. Melting analysis can also differentiate between specific and non-specific products (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

After the introduction of hydrolysis and hybridization probes, several other probe designs were adapted to, or created for, real-time PCR. Some of these probes increase in fluorescence when their conformation changes with hybridization and may function by hybridization and/or hydrolysis mechanisms depending on the reaction conditions. Hairpin probes, or "molecular beacons" can be monitored in real-time. These probes have a central sequence complementary to the DNA target and flanking ends complementary to each other. This configuration creates a hairpin at low temperatures. At higher temperatures in the presence of target, the probe hybridizes preferentially to the target. One end of the probe is labeled with a fluorophore and the other with a quencher so that when hybridized to the target, fluorescence increases. Hairpin probes use quenchers that release transferred energy as heat rather than light (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Hairpins can also be attached to the 5'-end of a PCR primer to generate self-probing amplicons during PCR. A fluorophore/quencher pair in the hairpin stem is linked to the primer with a blocking agent that prevents PCR read-through. The loop of the hairpin is complementary to the extension product of the primer so that once extension occurs, intramolecular hybridization separates the fluorophore/quencher pair and a larger hairpin is formed. This intra-molecular hybridization of self-probing amplicons is faster than the intermolecular hybridization of other probes. Hairpin primers can also be made without a blocking agent, allowing PCR read-through and incorporation of the stem-loop into the product. During PCR, the quencher/reporter pair is separated and fluorescence increases.

Before thermostable polymerases were used in PCR, thermal cyclers were unwieldy instruments with integrated fluidics to add fresh enzyme after each denaturation. Taq polymerase greatly reduced the engineering complexity of thermal cyclers, requiring only temperature cycling but not liquid handling. It did not take long before a variety of thermal cycling solutions appeared. Instruments progressed rapidly from laboratory oddities to mainstream commodities. Some early homemade examples changed the temperature of stationary reactions with flowing water or robotically transferred samples between constant temperature water baths (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). However, water has some drawbacks. Due to its large thermal mass a great amount of energy and time is required to heat or cool water to a specific temperature. In contrast, air has a very low thermal mass and was used in some early systems (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Many thermal cyclers now use Peltier elements and metal blocks for heating and cooling.

Today, PCR hardware and reagents are commonplace in research and diagnostic laboratories. The instruments have evolved to fill a variety of batch size and time-to-result needs. Thermal cycling concerns now focus on issues of speed, temperature uniformity, sample volume and increased throughput. Many thermal cycling solutions, heat-stable polymerases, and commercial PCR master mixes that include all components except primers and template DNA are available commercially.

A big step in PCR automation was connecting the amplification and detection stages to control PCR product contamination. Laboratories can be plagued by false positive results if products from a prior reaction find their way into a future reaction with the same primers. This contamination is usually controlled by separating pre- and post-amplification processes and careful attention to reaction preparation (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization). Another solution is to automate both amplification and detection in a closed-vessel system, eliminating PCR product exposure to the environment. The best solution is to amplify and analyze at the same time by real-time PCR and/or melting analysis.

The polymerase chain reaction (PCR) has become a fundamental tool in molecular research and clinical testing. Early evolution of the PCR process included adaptation to RNA, thermostable polymerases, automation, improvements in specificity and rapid temperature cycling. Perhaps the most significant advance is real-time PCR, combining both amplification and detection into one instrument as a superior solution for nucleic acid quantification. Real-time PCR is enabled by monitoring the reaction with double stranded DNA dyes or specific probes, including hydrolysis, hybridization, and conformation-sensitive probes. PCR product and probe melting analysis continues to improve in resolution, allowing greater sequence detail for genotyping and variant scanning. Microfluidic platforms and digital PCR are destined to find more applications in the future.

By measuring time, temperature, and fluorescence throughout PCR, real-time 3-dimensional spirals can be acquired and plotted. Software on commercial instruments usually only present selected data. For example, qPCR experiments only acquire fluorescence at one temperature each cycle. Typical melting analysis only acquires fluorescence from one melting curve at the end of amplification. Much more data is available during PCR, and it is likely that this additional data will find further use in the years to come.

Homogeneous monitoring of PCR is the method of choice for gene expression quantification and closed-tube genotyping. As a "gold standard", it has evolved from early conception to present-day reality. Future improvements will be focused on reducing cost and complexity (high resolution melting), decreasing reaction volumes (microfluidic PCR) and increasing throughput and sensitivity (digital PCR). These approaches will allow homogeneous monitoring of PCR to continue its evolution as a useful tool for many years to come (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

High Resolution Melting Analysis

Labeled primers became unnecessary with the development of saturating dsDNA dyes that could detect heteroduplexes throughout an amplicon. Single base genotyping within small amplicons required only two PCR primers and became the simplest method of genotyping. Because heterozygotes were easily identified by a change in melting curve shape, variant scanning was also enabled. Unlike other scanning techniques, high resolution melting does not require any physical processing or separations. It has been applied to cancer, many human genetic disorders and has been recently reviewed. In clinical diagnostics, greater sequence detail can be obtained if necessary with unlabeled probes or snapback primers (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Unlabeled probe genotyping requires only two standard primers and an unlabeled probe complementary to the sequence of interest. Unlabeled probes are generally designed to be 20-35 bases long and can match either the wild-type or variant sequence. Snapback primers incorporate the unlabeled probe as a 5'-extension on one of the primers. At low temperature the probe element snaps back onto its complementary sequence to form an intramolecular hairpin. With both unlabeled probes and snapback primers, different genotypes result in varying duplex stabilities which are easily resolved by high resolution melting. When unlabeled probes or snapback primers are used for genotyping, two melting transitions are generally observed, one for the full-length amplicon and the other for the probed region. This feature enables simultaneous variant scanning and genotyping in the same PCR reaction (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Microfluidic PCR

However, despite wide interest in microfluidic PCR, progress has slowed from a number of setbacks. Sample and reagent adsorption onto the reaction vessel surfaces can inhibit PCR and increase the risk of carryover contamination, due to the large surface area-to-volume ratios used. Solutions for sample adsorption have been explored with differing success. PCR efficiency in microfluidic PCR is often compromised, and the samples used for demonstration are often less complex targets such as plasmids, bacteria or previously amplified products at high concentrations.

There are two widely used designs for microfluidic PCR; stationary well and continuous flow. Stationary wells do not move the sample and operate in much the same way as traditional thermal cyclers. Both the sample and the device itself are heated/cooled through specific temperatures for PCR. The thermal mass is still large and thus more traditional cycling times are generally employed to perform the PCR. However, a matrix of microwells, mixing all combinations of X samples and Y targets can be obtained by microfluidics, greatly simplifying reaction preparation.

Continuous-flow microfluidic PCR has some advantages over PCR performed in stationary wells. Namely, by moving the sample through fixed temperature zones, this system can achieve faster cycling times due to the fact that only the sample needs to be heated and not the entire system. A comprehensive review of the various designs and techniques involved in microfluidic PCR is available (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).

Digital PCR

The total reaction volume of the many wells required for digital PCR make 96- or even 384-well plates unwieldy for high-throughput sample analysis. However, digital PCR performed on microfluidic PCR devices has been used for single-copy DNA droplet PCR, aneuploidy detection and absolute quantification of point variants.

Digital PCR improves detection specificity and sensitivity in samples with a large background of wild-type alleles compared to variant alleles and is the ultimate in allele quantification. The reduced cost associated with microfluidic devices may eventually make single-step, highly parallel individual PCR reactions for digital PCR affordable (Wittwer and Farrar, 2011 in PCR Troubleshooting and Optimization).]]>http://www.pcr-blog.com/2010/11/current-and-future-trends-in-pcr.htmlhttp://www.pcr-blog.com/2010/11/current-and-future-trends-in-pcr.htmlMon, 22 Nov 2010 12:51:46 GMTPCR TroubleshootingPCR ApplicationsReal-Time PCRPCR TechnologyThe strategies, tips and advice contained in this concise volume enable the scientist to optimize and effectively troubleshoot a wide range of techniques including PCR, reverse transcriptase PCR, real-time PCR and quantitative PCR. An essential book for anyone using PCR technology.

• Magic in Solution: An Introduction and Brief History of PCR
  Speaker: Carl Wittwer
  18 May 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET


• Obtaining Maximum PCR Sensitivity and Specificity
  Speaker: Cameron N. Gundry
  25 May 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET


• Significance of Controls and Standard Curves in PCR
  Speaker: Ian Kavanagh
  01 June 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET


• The MBD2-based Enrichment Approach for Analyzing DNA methylation
  Speaker: Chris Adams
  08 June 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET


• The MIQE Guidelines Uncloaked
  Speaker: Greg Shipley
  15 June 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET


• High Resolution Melting Analysis - Beyond the SNP
  Speaker: John Mackay
  22 June 2010 / 9am Pacific / 12pm Eastern / 5pm GMT / 6pm CET