Applications
PCR in Salmonella classification
PCR 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
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
Microfluidic Emulsion PCR
Microfluidic 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
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
High Resolution Melting Analysis
High 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
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
PCR Applications for Epigenetics Research
PCR 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
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
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