The new book on Real-Time PCR edited by Nick A. Saunders and Martin A. Lee will be available for dispatch within the next 2 or 3 weeks read more ...

Real-Time PCR: Advanced Technologies and Applications Edited by: Nick A. Saunders and Martin A. Lee ISBN: 978-1-908230-22-5 Publisher: Caister Academic Press Publication Date: July 2013 Cover: hardback

read more ...

The new book on Real-Time PCR in Food Science edited by David Rodríguez-Lázaro will be available for dispatch within the next 2 or 3 weeks read more ...

Real-Time PCR in Food Science: Current Technology and Applications Edited by: David Rodríguez-Lázaro ISBN: 978-1-908230-15-7 Publisher: Caister Academic Press Publication Date: January 2013 Cover: hardback

read more ...

Nick A. Saunders and Martin A. Lee present a new book on Real-Time PCR: Advanced Technologies and Applications
This essential manual provides both the novice and experienced user with an invaluable reference to a wide-range of real-time PCR technologies and applications and provides an overview of the theory of this increasingly important technique. Renowned international authors present detailed technical insights into the underlying principles, methods and practice of real-time PCR. The initial chapters cover the important aspects of real-time PCR including choosing an instrument and probe system, set-up, nucleic acid synthesis, sample extraction controls, and validation and data analysis. Further chapters provide a comprehensive overview of important real-time PCR methodologies such as quantification, expression analysis and mutation detection. This is complemented by the final chapters, which address the application of real-time PCR to diagnosis of infectious diseases, biodefence, veterinary science, food authenticity and molecular haplotyping. This timely and authoritative volume serves both as a basic introduction to real-time PCR and as a source of current trends and applications for those already familiar with the technology. The editors also aim to stimulate readers of all levels to develop their own innovative approaches to real-time PCR. An essential book for all laboratories using PCR read more ...

Real-Time PCR: Advanced Technologies and Applications Edited by: Nick A. Saunders and Martin A. Lee ISBN: 978-1-908230-22-5 Publisher: Caister Academic Press Publication Date: July 2013 Cover: hardback read more ...

Excerpt from a book review of Quantitative Real-time PCR in Applied Microbiology:

"reviews and illustrates the use of quantitative real-time PCR for a number of different purposes. It covers the basic process as well as the technology that has improved its performance, while also exploring the various scientific fields that use this technique routinely. It provides a complete description of what scientists need to design and perform a quantitative PCR ... useful to scientists in many different types of laboratories, including public health, environmental, clinical diagnostic, and food industry. It also can be useful to students and young investigators as well as experienced scientists. The authors clearly are familiar with the development and application of quantitative PCR and share their experience here ... This useful book is filled with valuable information for any laboratory using PCR to detect microbial agents and will serve as a resource for many years to come. " from Rebecca T. Horvat (University of Kansas, USA) writing in Doodys read more ...

Quantitative Real-time PCR in Applied Microbiology Edited by: Martin Filion ISBN: 978-1-908230-01-0 Publisher: Caister Academic Press Publication Date: May 2012 Cover: hardback "useful book ... filled with valuable information" (Doodys)

David Rodriguez-Lazaro presents a new book on Real-Time PCR in Food Science: Current Technology and Applications
Written by experts in the field, this book is an indispensable manual for scientists in the food industry. The first section provides an introduction to real-time PCR, discusses the use of PCR diagnostics in food science, describes the principles and methods of sample preparation, and covers the verification and control of PCR procedures. The eleven chapters in the second section cover the use of real-time PCR to detect various pathogens including Salmonella, Listeria, E. coli, Campylobacter, Yersinia, Staphylococcus, Clostridium, viruses and parasites. Also included is a chapter on the standardisation of real-time PCR methods in food microbiology. In the final section authors cover the use of real-time PCR for the analysis of genetically modified organisms, food allergens and for identification of animal or plant species. An invaluable book for anyone involved in food microbiology or the detection of foodborne pathogens and a recommended volume for all microbiology laboratories read more ...

Real-Time PCR in Food Science: Current Technology and Applications Edited by: David Rodriguez-Lazaro ISBN: 978-1-908230-15-7 Publisher: Caister Academic Press Publication Date: January 2013 Cover: hardback read more ...

The new book on Quantitative Real-time PCR in Applied Microbiology edited by Martin Filion will be available for dispatch within the next 2 or 3 weeks read more ...

Quantitative Real-time PCR in Applied Microbiology Edited by: Martin Filion ISBN: 978-1-908230-01-0 Publisher: Caister Academic Press Publication Date: May 2012 Cover: hardback

read more ...

from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

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.

from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

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.

from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

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.

Martin Filion (Department of Biology, Universite de Moncton, Canada) presents a new book on Quantitative Real-time PCR in Applied Microbiology
Written by experts in the field and aimed specifically at microbiologists, this volume describes and explains the most important aspects of current qPCR strategies, instrumentation and software. Renowned authors cover the application of qPCR technology in various areas of applied microbiology and comment on future trends. Topics covered include instrumentation, fluorescent chemistries, quantification strategies, data analysis software, environmental microbiology, water microbiology, food microbiology, gene expression studies, validation of microbial microarray data and future trends in qPCR technology. The editor and authors have produced an outstanding book that will be invaluable for all microbiologists. A recommended book for all microbiology laboratories read more ...

Quantitative Real-time PCR in Applied Microbiology Edited by: Martin Filion ISBN: 978-1-908230-01-0 Publisher: Caister Academic Press Publication Date: May 2012 Cover: hardback read more ...

from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

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.

Excerpt from a book review of PCR Troubleshooting and Optimization: The Essential Guide:
"The information is wholesome and appears to target both students and scientists knowledgeable in molecular applications. The comprehensive and comprehendible content indeed qualifies the text as an essential guide to the development, optimization and toubleshooting of PCR assays." from Christopher J. McIver writing in Aus. J. Med. Sci. (2011) 32: 68 read more ...

PCR Troubleshooting and Optimization: The Essential Guide Edited by: Suzanne Kennedy and Nick Oswald ISBN: 978-1-904455-72-1 Publisher: Caister Academic Press Publication Date: January 2011 Cover: hardback "an essential guide" Aus. J. Med. Sci.

from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

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).

from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

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.

from Wittwer CT and Farrar JS (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.

from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

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).

from Wittwer CT and Farrar JS (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.

from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

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.

from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

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).

from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

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).

from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

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

High resolution melting was first reported using a 5'-labeled primer for generation of fluorescence. Together with the development of high resolution melting instrumentation, high-quality, reproducible melting curves for variant scanning and genotyping became possible. High resolution instruments were necessary because standard real-time PCR instruments did not possess the precision necessary for distinguishing small differences between melting curves. However, the requirement for labeled primers and the limitation that variants had to be in the same melting domain as the primer were disadvantages.

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

Microfluidic PCR has the potential to amplify and quantify nucleic acids very quickly and reduce reagent demands and their associated cost. A wonderful concept, the professed "chip PCR" has yet to gain wide application in research or diagnostics. Chip PCR was first described in 1993. A microsystem for performing capillary electrophoresis was introduced around the same time and a system for incorporating both of these developments was demonstrated in 1996.

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

An interesting extension of microfluidic PCR is digital PCR. Digital PCR combines the amplification and quantification power of PCR with limiting dilution of template targets. This allows not only for the quantification of PCR products but also for quantification of rare initial nucleic acid targets, important in such areas as cancer and prenatal diagnostics. First demonstrated by dilution PCR, the method was later popularized on 96-well plates. By performing a dilution of the DNA pre-PCR, a single-template can be deposited in approximately every other well. Two probes are then used post-PCR for the determination of an allele ratio, one labeled with a green fluorescent dye and the other a red fluorescent dye. Simple comparison of well fluorescence determined the allele ratio.

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).

from Wittwer CT and Farrar JS (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.

from Wittwer CT and Farrar JS (2011) in PCR Troubleshooting and Optimization

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.