PCR Troubleshooting and Optimization
A new book on PCR 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
Further reading: PCR Troubleshooting and Optimization: The Essential Guide
Detection of Microbes in Water
Molecular techniques based on genomics, proteomics and transcriptomics are rapidly growing as complete microbial genome sequences are becoming available and advances are made in sequencing technology, analytical biochemistry, microfluidics and data analysis. While the clinical and food industries are increasingly adapting these techniques, there appear to be major challenges in detecting 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
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