The PCR Blog: The Polymerase Chain Reaction

qPCR 2010 Event

2009-11-25T08:55:00.000Z

April 7 - 9, 2010
Vienna, Austria Further information
Forum to exchange ideas, share experiences, and discuss the future of the perhaps most powerful analytical technology ever developed in the life sciences area, the quantitative real-time polymerase chain reaction (qPCR). Forty scientific talks from invited international scientists and diagnostic companies in the qPCR field who will show their latest research findings and newest technologies. Focus of the event will be the ongoing evolution of qPCR.
Suggested reading: PCR Books

PCR methods for the detection of Borrelia burgdorferi

2009-11-03T08:43:00.002Z

PCR is now a standard approach for direct detection of B. burgdorferi infection in tick vectors, host reservoirs, experimentally infected laboratory animals, and clinical specimens. PCR methods that are available for detection of B. burgdorferi DNA in various specimens include standard PCR, nested PCR, competitive PCR and real-time PCR (Wang et al., 2010).

The efficiency of a PCR assay is determined by several factors. Among these, the selection of an appropriate gene target and primer set for PCR amplification are the most important. In general, a PCR primer set yielding an amplicon of 100-300 bp is recommended, as it has high amplification efficiency under standard PCR conditions and can attentuate the effects of DNA fragmentation during sample processing.

Although PCR assays targeting numerous B. burgdorferi genes have been employed extensively in research settings, only a few of these genes have been widely utilized for PCR-based detection of B. burgdorferi s. l. These include the chromosomally encoded genes rrs, flaB, recA and p66, and the plasmid-encoded gene ospA (Wang et al., 2010).

Given that the number of spirochetes in infected tissues or body fluids of patients is generally very low, appropriate procedures for sample collection, transport and DNA preparation from clinical samples are critical for yielding reliable and consistent PCR results. Sensitivity of PCR assays may be reduced by degradation of the B. burgdorferi DNA during sample transport, storage, and processing. If the tissue is kept in BSK medium for over 24 h, some spirochetes may have migrated from the tissue to the culture medium. In this case, DNA prepared from both the tissue and the medium will increase the positivity rate of the PCR assay (Wang et al., 2010).

Clinical specimens collected from patients should be subjected to DNA extraction and PCR analysis shortly after collection; if this is not possible, specimens should be kept frozen until analysis. Studies on infected animal tissues suggest that PCR with DNA prepared from fresh frozen tissues has higher yields than that from paraffin-embedded, formalin-fixed tissues. As host DNA can interfere with PCR detection of B. burgdorferi in clinical and tick samples, an optimized DNA extraction procedure is essential to yield reliable PCR results for certain samples. Also, PCR inhibitors may be present in various biological materials (blood, urine, synovial fluid and cerebrospinal fluid) obtained from patients.

Real-time PCR has been used for detection, quantification and genotyping of pathogenic B. burgdorferi species in ticks and patients (Wang et al., 2010). The sensitivity of DNA extraction using manual or automated nucleic acid preparation systems is comparable for a variety of specimen types. Only a few genes have been used as targets in real-time PCR (e.g., ospA, rrs, hbb, recA) and these assays usually have used smaller fragments (100-200 bp) than those targeted in standard PCR tests. The sensitivity, specificity and reproducibility of real-time PCR and real-time quantitative PCR (qPCR) can be affected by a number of factors, including the properties of selected primers or probe, the quality of template DNA, the detection format (SYBR Green I-based or hybridization probe-based), and the amplification conditions (Wang et al., 2010).

References:
Wang, G., Aguero-Rosenfeld, M.E., Wormser, G.P. and Schwartz, I. (2010) Detection of Borrelia burgdorferi. In: Borrelia: Molecular Biology, Host Interaction and Pathogenesis (D. Scott Samuels and Justin D. Radolf, eds.). Caister Academic Press, Norfolk, UK.

Mackay, I.M. (2007) Real-Time PCR in Microbiology: From Diagnosis to Characterization. Caister Academic Press, Norfolk, UK.

SPUD qPCR Assay Confirms PREXCEL-Q Software's Ability to Avoid qPCR Inhibition

2009-10-02T16:48:00.002+01:00

Real-time quantitative polymerase chain reaction is subject to inhibition by substances that co-purify with nucleic acids during isolation and preparation of samples. Such materials alter the activity of reverse transcriptase (RT) and thermostable DNA polymerase enzymes on which the assay depends. When removal of inhibitory substances by column or reagent-based methods fails or is incomplete, the remaining option of appropriately, precisely and differentially diluting samples and standards to non-inhibitory concentrations is often avoided due to the logistic problem it poses.

To address this, the PREXCEL-Q software program was invented to automate the process of calculating the non-inhibitory dilutions for all samples and standards after a preliminary test plate has been performed on an experimental sample mixture. The SPUD assay was used to check for inhibition in each PREXCEL-Q-designed qPCR reaction. When SPUD amplicons or SPUD amplicon-containing plasmids were spiked equally into each qPCR reaction, all reactions demonstrated complete absence of qPCR inhibition. Reactions spiked with ~15,500 SPUD amplicons yielded a Cq of 27.39 +/- 0.28 (at ~80.8% efficiency), while reactions spiked with ~7,750 SPUD plasmids yielded a Cq of 23.82 +/- 0.15 (at ~97.85% efficiency).

This work demonstrates that PREXCEL-Q sample and standard dilution calculations ensure avoidance of qPCR inhibition.

from Gallup et al in SPUD qPCR Assay Confirms PREXCEL-Q Software's Ability to Avoid qPCR Inhibition

Further reading: PREXCEL-Q Software's Ability to Avoid qPCR Inhibition

Real-Time PCR book review

2009-08-13T09:16:00.002+01:00

I am pleased to provide the following excerpt from a recently published book review:
Real-Time PCR: Current Technology and Applications
"... written by international authors expert in specific technical principles and applications ... a useful compendium of basic and advanced applications for laboratory scientists. It is an ideal introductory textbook and will serve as a practical handbook in laboratories where the technology is employed." from Christopher J. McIver, Microbiology Department, Prince of Wales Hospital, New South Wales, Australia writing in Australian J. Med. Sci. 2009. 30(2): 59-60

Real-Time PCR: Current Technology and Applications

Edited by: Julie Logan, Kirstin Edwards and Nick Saunders
Published: 2009 ISBN: 978-1-904455-39-4
Price: GB £150 or US $310
A comprehensive guide to the most up-to-date real-time PCR technology and applications. The latest PCR platforms, fluorescent chemistries, validation software, data analysis, internal and external controls,clinical diagnostics, biodefense, RNA expression studies, validation of array data, mutation detection, food authenticity and legislation, NASBA, molecular halotyping. read more ...

PCR Troubleshooting

2009-06-18T12:01:00.001+01:00

An expert guide to PCR troubleshooting. The online guide covers problems with the template DNA, inadequate dNTPs, primer concentration, Taq concentration, Mg concentration, and KCl concentration. Most PCR problems are caused by one or other of these variables.

Find out the theory and possible solutions to these problems at PCR troubleshooting.

Cloning PCR Fragments

2009-06-08T15:31:00.002+01:00

Zuo and Rabie describe a new one-step "Quick Assemble" method of precisely and simultaneously joining multiple DNA fragments followed by intramolecular ligation. This technique of plasmid assembly and circularization can be used to construct new plasmids with any promoter, resistance gene marker, restriction site, or tag.

The new 2-day PCR-based method can be used to generate both circularized and linear final assembled products. The method makes the synthesis and assembly of large molecules in a quick and efficient way, while circumventing the use of DNA ligases. The ligase-free method also offers an additional improvement over the combination of long PCR and overlap extension PCR, and is expected to facilitate various kinds of complex genetic engineering projects that require precise in-frame assembly of multiple fragments, such as multiple site-direct mutagenesis and whole-DNA library gene shuffling.

from Zuo and Rabie in Curr. Issues Mol. Biol. 12: 11-16

Further reading: Assembly of DNA fragments into circular constructs

PCR Chip

2009-05-21T14:55:00.000+01:00

The polymerase chain reaction (PCR) has been widely used for amplification of DNA sequences. The PCR technique can be coupled with a variety of detection assays for the identification of a wide range of DNA targets. Reducing PCR to the microchip level is of interest for portable detection technologies and high-throughput, massively parallel analytical systems. The basic process of creating a miniaturized, microfluidic PCR chip and a simple method for thermally cycling this microchip during PCR amplification have been described (Herold and Rasooly, 2009. Lab-on-a-Chip Technology. ISBN: 978-1-904455-47-9) These methods can be broadly applied to a variety of microchip architectures, materials, and downstream analytical methods.

qPCR Web Resources

2009-04-28T14:48:00.001+01:00

A comprehensive list of qPCR Web Resources including qPCR machine manufacturers' websites, PCR web resources, and PCR news groups.

from Julie Logan, Kirstin Edwards and Nick Saunders in Real-Time PCR: Current Technology and Applications

Further reading: qPCR Web Resources

qPCR machines

2009-04-28T11:06:00.001+01:00

In weighing up the pros and cons of the different real-time PCR platforms or qPCR machines for your laboratory, factors to consider include: supported chemistries; multiplex capability for that chemistry; throughput; flexibility; format; easy-of-use and robust software package; reproducibility; speed; size; technical support; customer support and not least the cost, not only of the initial equipment outlay and servicing but also the associated cost of consumables and reagents.

The following qPCR machines are compared for various features to help you decide which instrument is most suitable for your needs.

Applied Biosystems: ABI 7300, ABI 7500, ABI 7500 Fast, ABI 7900 Fast HT with automation accessory, ABI StepOne
Roche: LightCycler 480, LightCycler 1.5, LightCycler 2.0
Stratagene: Mx4000, Mx3000P, Mx3005P
Cepheid: SmartCycler
Corbett: Rotor-Gene 6000
Eppendorf: Mastercycler ep realplex
BioRad: MiniOpticon, MyiQ, Opticon2, Chromo4, iQ5

qPCR machines. Part 6. Choosing a machine

2009-04-27T17:01:00.000+01:00

In weighing up the pros and cons of the different platforms for your laboratory, factors to consider include: supported chemistries; multiplex capability for that chemistry; throughput; flexibility; format; easy-of-use and robust software package; reproducibility; speed; size; technical support; customer support and not least the cost, not only of the initial equipment outlay and servicing but also the associated cost of consumables and reagents. It is also possible to 'try before you buy', most companies will provide a loan machine. It is wise to test a few of these once you have narrowed down your choice. User experiences should not be overlooked and there are now a number of useful websites and news groups where you can address you questions and queries.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

qPCR machines. Part 5

2009-04-27T16:58:00.000+01:00

Newly introduced qPCR machines include the Cepheid GeneXpert and Enigma Diagnostics Enigma ML instruments, which combine automated sample preparation and real-time PCR into a single integrated compact bench top instrument, which opens new avenues for real-time PCR in field-based and point-of-care environments. At the other end of the scale is the introduction of nanoliter high throughput quantitative PCR. The Biotrove OpenArray system allows 3072 33nl reactions on a microscope slide array format, which equates to a single operator performing 27,000 qPCR reactions in a working day. Another interesting development on the horizon is ultra fast real-time PCR chips capable of performing 40 cycles in under six minutes.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

qPCR machines. Part 4

2009-04-27T16:55:00.001+01:00

The majority of instrument manufacturers supply optimised real-time PCR mastermixes, these reagents benefit from being quality controlled, are easy to use, and usually offer reproducible and reliable results. However, for certain laboratories the additional cost can be prohibitive, however with an array of third party companies (e.g. Qiagen, Eurogentec, Invitrogen) now supplying real-time reagents, competition in this market should lead to reduced costs. Target specific kits for a range of applications are available from some manufacturers and other companies (e.g. Roche, Applied Biosystems, Qiagen). Additionally, Applied Biosystems offer Assays-on-Demand and Assays-by-Design services for SNP genotyping by real-time PCR. A number of companies now offer real-time PCR services from primer design right through to assay design and validation, quality assurance and other custom services.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

qPCR machines. Part 3

2009-04-27T16:53:00.003+01:00

Ideally, the analysis software supplied with the platform should be as user-friendly as possible but it is also important to check that the software can fully analyse results of the chosen probe chemistry. Some platforms and analysis software suites are biased towards certain chemistries. Some real-time instruments also have specific primer and probe design software that is either supplied with the hardware or available at extra cost. Such software can help simplify and speed up the assay design process and is optimised for that system and reagents. The LightCyclers also have specific relative quantification software that is designed to determine the exact relative nucleic acid concentration normalized to a calibrator sample. This software speeds up and greatly simplifies relative quantification.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

qPCR machines. Part 2

2009-04-27T16:53:00.001+01:00

Some platforms offer a low to medium throughput but are more flexible. Although the format may allow only 32 or even 16 samples, thermocycling times are faster and multiple runs can be performed thereby increasing the potential throughput. Performing multiple runs rapidly may be an advantage when several applications are employed which require different cycling parameters. The SmartCycler offers a different concept to real-time PCR of random access, which means independent programming of cycling parameters for 16 different assays. Each reaction vessel has its own element so that runs in available slots may be started any time, whilst other reaction sites are already in use. It is also modular allowing up to six units to be operated by a single computer. Others, such as the Chromo4 offers a modular design for maximum flexibility.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

qPCR machines. Part 1

2009-04-27T16:50:00.001+01:00

Several of the platforms employ a standard 96-well block format or interchangeable 384-well block and offer a medium to high throughput. An advantage of employing a block format is that standard PCR plates and tubes can be used and these tend to be cheaper than instrument-specific plastics (SmartCycler, Rotor-Gene) or glass capillaries (LightCyclers 1.5 & 2.0). Also, the LightCyclers 1.5 & 2.0 and SmartCycler require centrifugation to move sample into the reaction vessel and these alternative designs may not be suitable for all applications. For the highest throughput, the ABI 7900HT combines a 384-well plate format with an automation accessory, which allows for up to 84 plates to be loaded and unloaded, providing a throughput of >5000 wells per 8-hour day or 30,000 wells for end-point analysis only. The LightCycler 480 also offers a similar level of automation and high-throughput workflow capability. These high throughput instruments are ideal for dedicated laboratories where large batches of samples are run, with few different cycling parameters. An additional specification of a few platforms is the ability to perform gradient thermocycling, which can be very useful at the assay optimisation stage.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

Real-time PCR Chemistry: Choice of Platform

2009-04-09T08:25:00.001+01:00

Second, third and fourth generation real-time PCR platforms have been developed with improvements in multiplexing and increased throughput capabilities. The optical characteristics of a given platform clearly have an impact on the ability to multiplex and also determine which probe systems are compatible. In addition, the analysis software may also predetermine the appropriate chemistries.

Iit is important to point out that the platform and choice of fluorescent chemistry are strongly linked. Indeed some platforms are biased towards a particular probe system and whilst the optics permit different probe chemistries to be excited and detected, often the analysis software does not and the user is required to export the data to a spreadsheet program for detailed user analysis. For example, the Applied Biosystems platforms do not officially support any chemistries other than hydrolysis probes and SYBR Green and only support duplexing (Logan and Edwards, 2009). Therefore, the reporting chemistry required for an application should be strongly considered before a choice of platform is made.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

Real-time PCR Optics

2009-03-17T16:08:00.004Z

An integrated fluorimeter is required to detect and monitor the levels of fluorescence during the PCR process for real-time PCR and there is a range of options available for both the excitation light source and fluorescent emission detection. The light sources that cause fluorophore excitation can be classed as narrow- or broad-spectrum. If a broad-spectrum light source is employed (e.g. Mx4000, Mx3000P, Mx3005P, MyiQ, iQ5, ABI 7300, ABI 7500, LightCycler 480) then filters can be used to provide light tuned to the excitation spectrum of a specific individual fluorophore. Such a system provides the user with a wider choice of available fluorophores, although it is best to select those with good separation of their emission spectra.

A disadvantage of this optical system is that the light intensity passing through the filters can be limited and this could in theory limit the sensitivity of detection. There are currently two narrow-spectrum light sources used in real-time platforms, these can be light emitting diodes (LEDs) (e.g. LightCyclers 1.5 and 2.0, Opticon2, MiniOpticon, Chromo4, SmartCycler, Rotor-Gene, StepOne, Mastercycler ep) or laser (ABI 7900HT). The SmartCycler and Rotor-Gene have multiple LEDs (4 and 6 respectively) that excite at different wavelengths, providing a greater selection of fluorophores and giving these instruments capabilities similar to those of the broad-spectrum platforms above. The LightCyclers 1.5 & 2.0, Opticon2 and ABI 7900HT have single light source excitation, which ultimately limits the choice of fluorophores.

In general, the detectors used in real-time platforms are set to measure narrow bands of the spectrum, although filter sets that can be customised by the user are available for the iQ5, Mx4000, Mx3005P and Mx3000P, Chromo4. The number of detection channels that can be effective is dependent on the available range of excitation wavelengths. For example, if a single narrow range excitation source is available, one approach is to use fluorophores that are all excited to some extent in the same range and then to rely on software correction to deconvolute the light emitted from a given area of the spectrum, as was employed successfully with the now discontinued ABI 7700.

Another approach is demonstrated with the LightCycler 1.5, where a narrow-spectrum light source excites the fluorophores SYBR or fluorescein and emitted light is collected via three discrete optical detectors. Two of these detect long wavelength light emissions from fluorophores which are only minimally excited by the blue LED light source, which are instead excited using FRET technology.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

Real-time PCR Thermocycling

2009-03-10T08:58:00.001Z

The first component to consider in a PCR platform is the thermal engine. Successful thermal cycling is dependent on the accurate regulation of temperature in the sample vessels and the speed at which these target temperatures can be achieved. The majority of real-time platforms use advanced heating block technology based on the Peltier-effect, to actively transfer heat in and out of thin-walled plastic reaction vessels (e.g. ABI 7300, ABI 7500, ABI 7900HT, ABI Step One, Opticon 2, MiniOpticon, Chromo 4, Mx4000, Mx3000P, Mx3005P, Mastercycler ep, MyiQ, iQ5, LightCycler 480).

Peltier devices transfer heat from one side of a semiconductor to another. In general, blocks have significant mass and consequently a degree of thermal inertia. Furthermore, the plastic insulating layer between the reaction vessel and the heater produces an additional thermal lag. As a consequence of this, the temperature transitions are relatively slow and blocks must be very carefully designed to minimise well-to-well variation. Other advances on the Peltier-based technology include its combination with Joule, resistive or convective technology to give improved temperature control and performance across the block.

More recently has been the inclusion of patented Therma-Base technology, whose working principle is based on the evaporation and condensation of a working fluid in a thin vacuum cavity to accurately control well-to-well variation. Three platforms employ alternative heat exchange technologies which permit more rapid thermal ramp rates than blocks, resulting in significantly increased thermocycling speeds. These include a stationary turbulent air-heated glass capillary format (LightCyclers 1.5 and 2.0), a centrifugal air-heated plastic tube format (Rotor-Gene) and a high-thermal-conductivity ceramic heating plate plastic tube format (SmartCycler). For example, the time taken to equilibrate at 72°C using a Rotor-Gene is 0s compared to 15s with a standard 96-well peltier block, resulting in run times that are on average 50% faster. A recently published review (Logan and Edwards 2009) indicates that the LightCyclers 1.5 and 2.0 have the capacity to perform the fastest PCR and the Rotor-Gene has the smallest variation in temperature uniformity.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

Real-time PCR platforms

2009-02-17T16:30:00.002Z

A real-time PCR instrumentation platform consists of a thermal cycler, optics for both fluorescence excitation and emission collection, together with a computer and software for data acquisition and analysis.

A wide range of systems are available and these differ in design and level of sophistication. The variation of features includes: format, reaction vessels, emission and excitation wavelengths, throughput, level of control, chemistry, software, speed and applications. All real-time PCR machines have in common the ability to measure the accumulation of PCR product during the exponential phase of the reaction using online fluorescence monitoring, whether specific or non-specific and hence provide accurate data on initial starting copy numbers.

Amplification and detection are combined in a single step, therefore the process can occur in a single closed reaction vessel eliminating any need for post-PCR manual manipulations, and reducing the possibility of introducing contamination or variability.

Additional technical advantages include both qualitative and quantitative PCR, mutation analysis, multiplexing and high-throughput analysis. Although the fluorescence chemistries used in different platforms are similar, their mechanics and methodologies are wide ranging.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

History of Real-time PCR

2009-01-23T15:33:00.002Z

Originally, the simultaneous amplification and detection of specific DNA sequences in real-time was achieved by adding ethidium bromide (EtBr) to the PCR reaction so that the accumulation of PCR product could be visualized at each cycle. When EtBr is bound to double-stranded DNA and excited by UV light it fluoresces, therefore an increase in fluorescence in the reaction indicates positive amplification. Soon afterwards real-time PCR product quantitation or "kinetic PCR" was achieved by continuously measuring the increase in EtBr intensity during amplification with a charge-coupled device camera. By creating amplification plots of fluorescence increase versus the cycle number the kinetics of EtBr fluorescence accumulation during thermocycling was directly related to the starting number of DNA copies. When a greater number of target molecules are present fewer cycles are needed to produce a detectable signal.

Kinetic monitoring also provided a means whereby the efficiency of amplification under different conditions could be determined, providing for the first time insight into the fundamental PCR processes. The principle underlying quantitative real-time PCR can be defined as the monitoring of fluorescent signal from one or more PCR reactions, cycle-by-cycle, to completion, where the amount of product produced during the exponential amplification phase can be used to determine the amount of starting material.

The use of EtBr was not ideal since EtBr binds non-specifically to DNA duplexes and non-specific amplification products, such as primer–dimers, can contribute to the fluorescent signal and result in quantification inaccuracies. Subsequent refinements, the most significant of which was the introduction of fluorogenic probes to monitor product accumulation, added a greater element of specificity to real-time PCR and provided greater quantitative precision and dynamic range than previous methods.

These significant advances to the basic PCR technique led to the development of a new generation of PCR platforms and reagents, which allowed simultaneous amplification and quantification of specific nucleic acid sequences cycle-by-cycle. The first commercial platform to become available was the Applied Biosystems ABI Prism 7700 Sequence Detection System, followed by the Idaho Technology LightCycler (later manufactured and sold by Roche Diagnostics). Both of these platforms utilized fluorogenic chemistry and like any real-time PCR platform, they basically consist of a thermal cycler with an integrated optical detection system, which can heat, cool, detect and report. New and improved models have now superseded these two instruments and several other manufacturers have introduced their own real-time PCR platforms.

Real-time PCR offers many advantages over traditional PCR, including the amplification and detection in an integrated system, fluorescent dyes/probes allowing constant reaction monitoring, rapid cycling times (20-40 mins for 35 cycles), high sample throughput (200 to 5000 samples/day), low contamination risk due to sealed reactions, increased sensitivity, detection across a broad dynamic range of 10 - 1010 copies, reproducibility, quantification of results, and software driven operation.

from Logan and Edwards (2009) in Real-Time PCR: Current Technology and Applications

Bibliography:

Real-Time PCR Data Analysis

2009-01-19T14:30:00.004Z

Quantitative real-time RT-PCR (qRT-PCR) is widely and increasingly used in any kind of mRNA quantification, because of its high sensitivity, good reproducibility and wide dynamic quantification range. While qRT-PCR has a tremendous potential for analytical and quantitative applications, a comprehensive understanding of its underlying principles is important. Beside the classical RT-PCR parameters, e.g. primer design, RNA quality, RT and polymerase performances, the fidelity of the quantification process is highly dependent on a valid data analysis.

The software provided with real-time PCR instruments allows several types of data analysis:

normalisation of the raw data
measurement of the cycle number at which any increase in the fluorescence within each reaction vessel reaches significance
the data are used in conjunction with the results from internal or external standards to estimate the original number of template copies
melting curves are transformed to provide plots of –dF/dT against T (F = fluorescence and T= temperature) in which a peak (melting peak) occurs at the equilibrium temperature for each duplex

In general instrument specific software is easy to use and allows rapid and reproducible data analysis. In addition to the bundled software a range of third party utilities is available to improve the flexibility of real-time PCR data analyses.

from M.W. Pfaffl, J. Vandesompele and M. Kubista in Real-Time PCR: Current Technology and Applications

Further reading: Real-Time PCR

PCR Microchips: applications in forensic, clinical and biological fields

2009-01-14T13:56:00.002Z

A novel circular ferrofluid driven microchip has been developed for rapid polymerase chain reaction (PCR). A closed-loop circular channel was fabricated on one microchip and the PCR mixture together with a small ferrofluid plug was injected into the loop. An external magnet is used to drive the ferrofluid plug, which in turn propels the PCR mixture to move around and flow continuously through three pre-set temperature zones.

Parameters of PCR, such as incubation time, temperatures and number of cycles, can be fully controlled and adjusted. To improve throughput, a multi-loop ferrofluid driven microchip was also developed by designing a series of concentric circular channels on one microchip and the magnet enabled simultaneous actuation of DNA samples in all the channels. High reproducibility was achieved for different channels in the same run and for the same channels in consecutive runs.

The circular ferrofluid-driven PCR microchips combine the cycling flexibility of the chamber PCR and the quick temperature transitions associated with the continuous flow (CF) PCR. Most importantly, the small footprint and simultaneous actuation make it the right candidate for parallel PCR analysis. The simple, reliable and high-throughput PCR microchips will find wide applications in forensic, clinical and biological fields.

from Sun et al in Lab-on-a-Chip Technology (2009) Herold KE and Rasooly A (eds) Published by Caister Academic Press

Further reading:

Clinical microbiology and real-time PCR

2009-01-12T15:36:00.002Z

The first PCR methods to be described for clinical microbiology utilized gel electrophoresis for the detection of PCR amplification products (Real-Time PCR in Microbiology: From Diagnosis to Characterization). Although these assays proved useful, their specificity and sensitivity was compromised by this rather cumbersome end-point detection method. Specificity of detection could be improved by incorporating a solid phase hybridization such as Southern blotting; however, this was labour intensive and time consuming requiring further manipulation of the PCR product.

Detection of PCR products by solid phase hybridization also limited the numbers of samples that could be processed, and the methods used were difficult to standardize between laboratories. The overall time taken to produce a result from a PCR assay could be two or three days and the test required a significant level of technical skill limiting the use of PCR to specialized laboratories. The introduction of enzyme-linked hybridization probe formats (PCR-ELISA) for the detection of amplification products did improve the detection process; however, they still required manipulation of the amplification products following PCR. Manipulation of the amplified product increases the likelihood of contaminating subsequent PCR reactions leading to false positives a phenomenon known as amplicon carryover.

PCR-ELISA facilitated the introduction of quantitative PCR (QPCR) assays; however, the range and accuracy of quantitation was limited. The more recent introduction of real-time platforms for PCR has revolutionized molecular diagnostic detection methods in clinical microbiology. These closed tube systems virtually eliminate the risk of amplicon carryover because the samples are not opened following thermal cycling. Many of these new platforms process samples more rapidly than conventional block-based thermal cyclers making pathogen testing much more rapid. In addition, the ability to monitor the reaction in real-time provides results immediately after cycling and facilitates quantitation of the original target sequence over many orders of magnitude. Realtime platforms can differentiate between several closely related sequences within the same reaction therefore assays can be multiplexed to detect a range of pathogens within the same tube. Many of the assays described to date have utilized the Idaho LightCycler or the Roche LightCycler instrument. Some of the other commonly used platforms for real-time PCR are the Applied Biosystems ABI Prism 7000, 7500, and 7900 Sequence Detection Systems, and the Cepheid Smart Cycler.

The real-time PCR method has been applied in virtually all areas of clinical microbiology and has proven useful in a wide range of applications.

Quality control has an important role in the implementation of molecular diagnostic testing for the diagnosis of infectious disease. Quality control encompasses measures such as the inclusion of appropriate positive, negative, and inhibition controls in assay runs. The results of positive controls should be monitored over time to ensure the assay is performing consistently and that inter-assay reproducibility remains high. External quality control schemes will play a very crucial role to ensure high standards in molecular diagnostic in the future.

The first external quality control scheme to be developed was the European Union Quality Control Concerted Action for Nucleic Acid Amplification in Diagnostic Virology. This temporary entity has been superseded by Quality Control for Molecular Diagnostics, a non-profit organization for the standardization and quality control of molecular diagnostics and genomic technologies. This organization sends out proficiency panels of simulated clinical samples containing a wide range of viral and bacterial pathogens for molecular diagnostic assays. Over 100 laboratories from more than 60 countries regularly participate in the program which is endorsed by the European Society for Clinical Virology and the European Society for Microbiology and Infectious Disease. Laboratories providing molecular diagnostic testing should participate in this scheme to ensure quality of testing.

The introduction of real-time PCR methods in clinical microbiology has improved the detection of infectious disease agents and led to improvements in patient management and care. In the future new developments in real-time molecular diagnostics will lead to further benefits to the patient consolidating the role of real-time PCR as an essential tool in the clinical microbiology laboratory.

from Andrew David Sails in Real-Time PCR: Current Technology and Applications

Further reading:

Real-Time PCR review

2009-01-12T14:53:00.003Z

The Polymerase chain reaction (PCR), which was first described in 1988, is one of the most important methodological developments in molecular biology in the last three decades. In a three-step reaction it is possible to exponentially increase the amount of nucleic acid from complex biological material, which can then be used for further analysis. A further advancement of this method is real-time (RT) PCR, which allows the continuous monitoring of nucleic acid amplification by fluorescent changes measured in real time. There are already numerous books about RT-PCR on the market, all of which stress the importance of this technology nowadays. In fact, this book is considered the second edition of the previously published book Real-Time PCR: An Essential Guide by the same editors in 2004.

The present book comprises 17 chapters, of which the first seven chapters provide an overview of the technology itself, and the following 10 chapters deal with it's applications. The first part of the book contains all information necessary for any scientist working on RT-PCR. The theory behind this technology, different platforms, different chemistries, and data analysis software are well explained. Even the aspect of validation is covered, which is of utmost importance when using this method in, for instance, the pharmaceutical industry.

The second part, which is introduced through Chapter 8 Introduction to the Applications of Real-Time PCR comprises some of the most important applications of the described technology. These applications include mRNA expression analysis, microarray data validation, mutation detection, fungal infections diagnosis, to mention a few. The most comprehensive chapter is Chapter 13 Application in Clinical Microbiology, in which large numbers of microorganisms are mentioned that can be readily identified by RT-PCR. However, the author of this review misses out the difficulties encountered, when a group of microorganisms, such as mycoplasmas, are the target.

Each chapter starts with an abstract to introduce the theme of the chapter. Most reference lists at the end of each chapter are extensive and provide additional sources for the interested reader. Sometimes cross references would be helpful, such as in the case of Locked Nucleic Acid, which is mentioned in Chapter 3, but explained in Chapter 9. Figures are numerous in most chapters, but the accompanying explanations are not always sufficient. Despite these minor criticisms, the editors, who are all affiliated at the Health Protection Agency, Centre of Infections, in London, UK, succeeded in providing a comprehensive overview of the RT-PCR technology, which is as up-to-date as a book can be in such a rapidly advancing field, considering the time span taken to publish such a book. The price for this hardcover book is comparable to that of other scientific books, which is unfortunately on the high side.

from Mareike Viebahn, in Current Issues in Molecular Biology

Further reading: Real-Time PCR: Current Technology and Applications

Other books of interest:

SNP Detection by Real-Time PCR

2009-01-07T17:18:00.002Z

The methods used to verify the identity of the amplicon(s) produced in real-time PCR are also sufficiently powerful to detect small variations between sequences. Variations in sequence, including single nucleotide polymorphisms (SNPs) have been successfully identified in real-time PCR assays. One common approach to the detection of sequence variation is to compare melting curves. In general, the effect of base substitutions on the melting kinetics of PCR products is too small to be detected reliably (if at all). However, heteroduplexes of relatively long amplicons differing by a SNP can be distinguished from the homoduplexes on the basis of their melting curves.

The melting curves of short fluorescent probes can be used to distinguish between amplicons. This method is sensitive to SNPs, which usually cause a shift in the melting peak of several degrees. A common alternative to the melting curve approach is to use hydrolysis (TaqMan) probes. The efficiency of the 5'-3' endonuclease reaction is greatly impaired when a well-designed probe mismatches its target sequence by even a single base. The detection of mutations by real-time PCR is discussed by Lyon et al Mutation Detection and by Pont-Kingdon Molecular Haplotyping. Although the melting curve and hydrolysis probe methods for mutation analysis are widely used they are only able to detect sequences that represent a large proportion of the population. A quantitative real-time ARMS method can be used (see Lyon et al). ARMS assays are designed to detect the emergence of significant sequence mutants within a background that remains mainly of the parent type.

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