http://www.pcr-blog.com/index.htmlPCR Blog RSS feedenCopyright 2010 Hugh2010-06-16T16:04:19+01:00 hourly 1 2000-01-01T12:00+00:00 Wed, 23 Jun 2010 12:21:33 +0100Applications2010-06-16T16:04:19+01:00http://www.pcr-blog.com/files/pcr-salmonella.html#unique-entry-id-15http://www.pcr-blog.com/files/pcr-salmonella.html#unique-entry-id-15PCR in sub-species level Salmonella classification
from Burkhard Malorny, Elisabeth Hauser and Ralf Dieckmann writing in Salmonella: From Genome to Function

Salmonellae form a complex group of bacteria consisting of two species, 6 subspecies and more than 2,500 serovars (serotypes). Salmonella identification below species level is most often limited to phenotypic typing methods such as biochemical and serological identification, which are costly, time-consuming and do not always reflect the evolution of Salmonella groups. Newer methods for Salmonella typing and subtyping include (multiplex-) PCR-based methods. In recent years further molecular typing technologies were evaluated for this purpose. A recent review discusses some of these emerging technologies. These new techniques promise significant advantages compared to traditional culture-based methods with respect to speed, ease of use, reliability and automation.

Further reading: Salmonella: From Genome to Function]]>TechnologyApplications2010-06-15T15:01:54+01:00http://www.pcr-blog.com/files/microfluidic-emulsion-pcr.html#unique-entry-id-14http://www.pcr-blog.com/files/microfluidic-emulsion-pcr.html#unique-entry-id-14Microfluidic Emulsion PCR
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]]>ApplicationsReal-Time PCRqPCRReal-Time qPCR2010-06-15T14:58:33+01:00http://www.pcr-blog.com/files/high-resolution-melting-analysis.html#unique-entry-id-13http://www.pcr-blog.com/files/high-resolution-melting-analysis.html#unique-entry-id-13High Resolution Melting Analysis
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.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide
]]>Applications2010-06-15T14:55:19+01:00http://www.pcr-blog.com/files/pcr-epigenetics.html#unique-entry-id-12http://www.pcr-blog.com/files/pcr-epigenetics.html#unique-entry-id-12PCR Applications for Epigenetics Research
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.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]>MIQEqPCRReal-Time PCR2010-06-15T14:53:49+01:00http://www.pcr-blog.com/files/miqe.html#unique-entry-id-11http://www.pcr-blog.com/files/miqe.html#unique-entry-id-11The MIQE Guidelines Uncloaked
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. An expanded explanation of the guideline items on how those requirements might be met should be consulted prior to publication.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]>qPCRReal-Time PCRData Analysis2010-06-15T14:51:18+01:00http://www.pcr-blog.com/files/qpcr-data-analysis.html#unique-entry-id-10http://www.pcr-blog.com/files/qpcr-data-analysis.html#unique-entry-id-10qPCR Data Analysis: Unlocking the Secret to Successful Results
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. Data analysis aspects include 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]]>TechnologyReal-Time PCRInstrumentationqPCR2010-06-15T14:48:57+01:00http://www.pcr-blog.com/files/real-time-pcr-instrumentation.html#unique-entry-id-9http://www.pcr-blog.com/files/real-time-pcr-instrumentation.html#unique-entry-id-9Real-Time PCR Instrumentation: An Instrument Selection 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 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. 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 instruments 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). Key features differentiate Real-Time PCR instruments, and various criteria should be considered when selecting the instrument that best fits a specific scientist's research needs.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]>OptimizationTechnologyTroubleshootingReal-Time PCR2010-06-15T14:45:32+01:00http://www.pcr-blog.com/files/rt-pcr-optimization.html#unique-entry-id-8http://www.pcr-blog.com/files/rt-pcr-optimization.html#unique-entry-id-8RT-PCR Optimization Strategies
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. Optimization of the whole RT-PCR workflow is important and recommendations to reduce experimental variance and produce more reproducible and reliable results should be followed.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]>Optimization2010-06-15T14:42:10+01:00http://www.pcr-blog.com/files/pcr-sensitivity-specificity.html#unique-entry-id-7http://www.pcr-blog.com/files/pcr-sensitivity-specificity.html#unique-entry-id-7Obtaining Maximum PCR Sensitivity and Specificity
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. Addressing all of these factors will aid the investigator in designing high quality PCR assays.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]>TechnologyTroubleshooting2010-06-15T12:17:31+01:00http://www.pcr-blog.com/files/pcr-controls.html#unique-entry-id-6http://www.pcr-blog.com/files/pcr-controls.html#unique-entry-id-6Significance of Controls and Standard Curves in PCR
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. There are strategies for preparing both negative and positive controls for PCR, when they should be employed and how to interpret the information they provide. Standard curves are vital for determining the initial starting amount of the target template and for assessing assay efficiency, precision, sensitivity, and dynamic range. It is important to know how to prepare standards, interpret standard curve and troubleshoot inefficient qPCR reactions.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]>Troubleshooting2010-06-15T12:09:51+01:00http://www.pcr-blog.com/files/inhibitors-of-pcr.html#unique-entry-id-5http://www.pcr-blog.com/files/inhibitors-of-pcr.html#unique-entry-id-5Difficult Templates and Inhibitors of PCR
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, there are strategies for detecting, avoiding and/or eliminating inhibition during reverse transcription, PCR, RT-PCR and qPCR. Also there are strategies to amplify difficult templates and optimize reverse transcription reactions.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]>HistoryTechnology2010-06-15T12:05:33+01:00http://www.pcr-blog.com/files/brief-history-of-pcr.html#unique-entry-id-4http://www.pcr-blog.com/files/brief-history-of-pcr.html#unique-entry-id-4A Brief History of PCR
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. A recent review by Wittwer and Farrar discusses the origins of PCR, its early evolution 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. 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.

Read more: PCR Troubleshooting and Optimization: The Essential Guide]]>TechnologySeminar2010-06-07T17:21:37+01:00http://www.pcr-blog.com/files/miqe.html#unique-entry-id-3http://www.pcr-blog.com/files/miqe.html#unique-entry-id-3
Next up we have MIQE Guidelines Uncloaked, in which Greg Shipley will give you the inside track on the requirements you need to satisfy to make sure your PCR results are suitable for publication. You'd be mad to miss it.

This event goes out live tomorrow (Tue 8th June) at 9am Pacific / 12pm Eastern / 5pm BST (UK) / 6pm CET. Click here to secure one of the remaining places on this live event..

You can also click here to take a look at our archive for this series, which now contains:

Magic in Solution: An Introduction and Brief History of PCR
Speaker: Carl Wittwer

Obtaining Maximum PCR Sensitivity and Specificity
Speaker: Cameron N. Gundry Attendence: 125

Significance of Controls and Standard Curves in PCR
Speaker: Ian Kavanagh]]>Home2010-05-13T15:30:16+01:00http://www.pcr-blog.com/files/getting-the-most-out-of-pcr.html#unique-entry-id-2http://www.pcr-blog.com/files/getting-the-most-out-of-pcr.html#unique-entry-id-2Getting The Most Out of PCR", is being broadcast by the popular life science blog, Bitesize Bio. Bitesize Bio is headed by Nick Oswald and Suzanne Kennedy, co-editors of the forthcoming book "PCR Troubleshooting and Optimization".

The series lineup includes many of the authors from this book and commences on May 18 with a talk from LightCycler co-inventor, Carl Wittwer, entitled "Magic in Solution: An Introduction and Brief History of PCR". This will be a great learning experience with an opportunity to ask questions and learn from experts and pioneers in the PCR field. The full program is shown below.

Click here to book your place on these excellent events.

• 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

Recommended reading: PCR publications]]>TroubleshootingOptimizationControl2010-03-29T16:54:02+01:00http://www.pcr-blog.com/files/pcr-troubleshooting.html#unique-entry-id-1http://www.pcr-blog.com/files/pcr-troubleshooting.html#unique-entry-id-1PCR Troubleshooting and Optimization has been announced by Caister Academic Press. Included in the book is: Strategies for preparing effective controls and standards for PCR, when they should be employed and how to interpret the information they provide. The significance of optimization for efficiency, precision and sensitivity of PCR methodology and essential guidance on how to troubleshoot inefficient reactions. Design and optimization techniques, the use of appropriate controls, the significance of standard curves and the principles and strategies required for effective troubleshooting. The importance of sample preparation and quality, primer design, controlling inhibitors, avoiding amplicon and environmental contamination, optimizing reagent quality and concentration, and modifying the thermal cycling protocol for optimal sensitivity and specificity.

Further reading: PCR Troubleshooting and Optimization: The Essential Guide]]>TechnologyApplicationsqPCREnvironmentalReal-Time PCR2010-03-12T14:20:38+00:00http://www.pcr-blog.com/files/pcr-detection-microbes.html#unique-entry-id-0http://www.pcr-blog.com/files/pcr-detection-microbes.html#unique-entry-id-0detecting health-related microbes in source and treated drinking waters. This is due in part to the low density of pathogens in water, necessitating significant processing of large volume samples. Quantitative PCR is a state-of-the-art technique available for pathogen detection and characterization from water.

Although quantitative PCR is almost 15 years old, only recently has it become a tool for diagnostic purposes in water microbiology. Conventional PCR and its variations largely give qualitative results (MPN-PCR being an exception) and are most useful when presence-absence of the target is to be noted. Since the product is measured at the end of the PCR, where the amount of amplicon (product) in a given reaction tube is likely to have been affected by saturation effects of excess amplicons or poorly optimized reactions, the yield of amplicon does not relate to the original starting concentration. Furthermore, a second step is always required for verification of the product. Because there is a quantitative relationship between amount of starting target and amount of PCR product during the exponential phase of the PCR process, if the yield of amplicons are made in the exponential or initial linear phases of amplification, which is the case in qPCR, then the data obtained can provide a quantitative relationship to the starting concentration.

In qPCR, fluorescent dyes and probes are generally used in addition to regular PCR primers, thus allowing for in situ assay of the targeted amplicon. With increasing cycles of PCR, the increase in target is directly quantified by an increase in fluorescence that is emitted by increased intercalation of fluorescent dye or hybridization of fluorescent oligonucleotide probe(s) to the target. These techniques are not "quantitative" in the strictest sense, as they measure a kinetic reaction. Often called "real time" or kinetic PCR, they do not measure the reaction as it occurs, but measure the results of the reaction in a pause between cycles. Perhaps the most correct descriptor is Kinetic PCR, but that term has not been adopted in molecular microbiology.

Further reading: Environmental Microbiology: Current Technology and Water Applications]]>