Real-Time PCR
Real-time PCR has removed many of the limitations of standard end-point PCR and since its introduction in the mid-1990s there has been an explosion both in the number of publications and available instrumentation describing real-time PCR applications across many disciplines. Real-time PCR (RT-PCR) technology is highly flexible and many alternative instruments and fluorescent probe systems have been developed recently. The decreased hands-on time, increased reliability and improved quantitative accuracy of RT-PCR methods have contributed to the adoption of RT-PCR for a wide range of new applications.
The development of instruments that allowed real-time monitoring of fluorescence within PCR reaction vessels was a significant advance. The technology is flexible and many alternative instruments and fluorescent probe systems are available. RT-PCR assays can be completed rapidly since no manipulations are required after the amplification. Identification of the amplification products by probe detection in real-time is highly accurate compared with the traditional PCR method of size analysis on gels. Analysis of the progress of the reaction allows accurate quantification of the target sequence over a very wide dynamic range, provided suitable standards are available. Further investigation of the RT-PCR products within the original reaction mixture using probes and melting analysis can detect sequence variants including single base mutations. RT-PCR has found applications in many branches of biological science. Applications include gene expression analysis, the diagnosis of infectious disease and human genetic testing. Due to their fluorimetry capabilities, these real-time machines are also compatible with alternative amplification methods such as NASBA, provided a fluorescence end-point is available.
The introduction of RT-PCR assays to the clinical microbiology laboratory has led to significant improvements in the diagnosis of infectious disease. The technology has applications in clinical bacteriology, parasitology and virology. There are few areas of clinical microbiology which remain unaffected by this new method. It has been particularly useful to detect slow growing or difficult to grow infectious agents.Its greatest impact is probably its use for the quantitation of target organisms in samples. The ability to monitor the PCR reaction in real-time allows accurate quantitation of target sequence over at least six orders of magnitude. The closed-tube format which removes the need for post-amplification manipulation of the PCR products also reduces the likelihood of amplicon carryover to subsequent reactions reducing the risk of false-positives. Standardisation of assay protocols for use in diagnostic clinical microbiology and external quality control schemes is required to ensure quality of testing.
Further reading:
Real-Time PCR: Current Technology and Applications
Real-Time PCR in Microbiology: From Diagnosis to Characterization
The development of instruments that allowed real-time monitoring of fluorescence within PCR reaction vessels was a significant advance. The technology is flexible and many alternative instruments and fluorescent probe systems are available. RT-PCR assays can be completed rapidly since no manipulations are required after the amplification. Identification of the amplification products by probe detection in real-time is highly accurate compared with the traditional PCR method of size analysis on gels. Analysis of the progress of the reaction allows accurate quantification of the target sequence over a very wide dynamic range, provided suitable standards are available. Further investigation of the RT-PCR products within the original reaction mixture using probes and melting analysis can detect sequence variants including single base mutations. RT-PCR has found applications in many branches of biological science. Applications include gene expression analysis, the diagnosis of infectious disease and human genetic testing. Due to their fluorimetry capabilities, these real-time machines are also compatible with alternative amplification methods such as NASBA, provided a fluorescence end-point is available.
The introduction of RT-PCR assays to the clinical microbiology laboratory has led to significant improvements in the diagnosis of infectious disease. The technology has applications in clinical bacteriology, parasitology and virology. There are few areas of clinical microbiology which remain unaffected by this new method. It has been particularly useful to detect slow growing or difficult to grow infectious agents.Its greatest impact is probably its use for the quantitation of target organisms in samples. The ability to monitor the PCR reaction in real-time allows accurate quantitation of target sequence over at least six orders of magnitude. The closed-tube format which removes the need for post-amplification manipulation of the PCR products also reduces the likelihood of amplicon carryover to subsequent reactions reducing the risk of false-positives. Standardisation of assay protocols for use in diagnostic clinical microbiology and external quality control schemes is required to ensure quality of testing.
Further reading:
Real-Time PCR: Current Technology and Applications
Real-Time PCR in Microbiology: From Diagnosis to Characterization
Labels: diagnostics, PCR, qPCR, real-time PCR, RT-PCR
Epigenetics
The field of epigenetics has gained great momentum in recent years and is now a rapidly advancing field of biological and medical research. Epigenetic changes play a key role in normal development as well as in disease.
The term epigenetics was coined by the developmental biologist Conrad Waddington to describe "the interactions of genes with their environment which bring the phenotype into being". This was recognition of the fact that it is not only the DNA sequence that determines the phenotype. All cells of a multicellular organism carry the same genetic material coded in their DNA sequence, but cells obviously display a broad morphological and functional diversity. Epigenetics is the branch of biology that studies the additional layers of information, in addition to the bare genomic sequence, that dramatically extend the information potential of the genetic code. Epigenetics, therefore, is the study of heritable changes in the cellular state that are not caused by changes in the nucleotide sequence of the DNA. Epigenomics is a new science unifying epigenetics and genomics and studying epigenetic modifications at high-throughput and/or on a whole genome level.
DNA methylation is the only genetically programmed DNA modification in mammals and probably the best studied epigenetic modification. Cytosine methylation at CpG positions of the DNA sequence is one of the hallmarks of epigenetic gene silencing. During evolution, CpG rich regions, so-called CpG islands, have been established as prominent features of promoter regions of genes. Whereas most regions of the genome are constantly methylated these elements are mainly kept free of methylation thereby facilitating the establishment of an open chromatin structure and of initiation of transcription. Besides its role in the regulation of genes, DNA methylation silences repetitive elements and appears to be important for the stability of the mammalian genome.
DNA-associated histone proteins play an important role in gene regulation within the mammalian genome. Various covalent chemical modifications of the histones are possible and can directly affect various DNA-templated processes such as transcription.
Polycomb and Trithorax group proteins are important epigenetic regulators of homeotic genes. They play a broad role in cell differentiation, which is exerted through the direct control of hundreds of transcription factors as well as important signaling proteins and morphogens. Polycomb silencing is a dynamic process intimately dependent on histone modifications and balanced by antagonistic action of Trithorax proteins.
In eukaryotic cells, the majority of transcribed RNAs are non-coding RNAs (ncRNAs). It is evident that ncRNAs are functionally involved in many biological processes, such as proliferation, differentiation and development. NcRNAs function as regulators of gene expression on various levels, including chromatin modification, transcription, RNA modification, RNA splicing, RNA stability and translation.
Genetic and epigenetic mechanisms contribute to the development of human tumors. Research into the differences between normal and cancer epigenomes, and the knowledge gained from single gene and large-scale epigenome analyses provide important information relevant to for gene discovery, therapeutic applications, and building a mechanism-based model of human tumorigenesis.
DNA methylation, chromatin structure, Polycomb proteins and non-coding RNAs are areas of current research and part of the fascinating and fast moving field of epigenetics.
Further reading: Epigenetics
The term epigenetics was coined by the developmental biologist Conrad Waddington to describe "the interactions of genes with their environment which bring the phenotype into being". This was recognition of the fact that it is not only the DNA sequence that determines the phenotype. All cells of a multicellular organism carry the same genetic material coded in their DNA sequence, but cells obviously display a broad morphological and functional diversity. Epigenetics is the branch of biology that studies the additional layers of information, in addition to the bare genomic sequence, that dramatically extend the information potential of the genetic code. Epigenetics, therefore, is the study of heritable changes in the cellular state that are not caused by changes in the nucleotide sequence of the DNA. Epigenomics is a new science unifying epigenetics and genomics and studying epigenetic modifications at high-throughput and/or on a whole genome level.
DNA methylation is the only genetically programmed DNA modification in mammals and probably the best studied epigenetic modification. Cytosine methylation at CpG positions of the DNA sequence is one of the hallmarks of epigenetic gene silencing. During evolution, CpG rich regions, so-called CpG islands, have been established as prominent features of promoter regions of genes. Whereas most regions of the genome are constantly methylated these elements are mainly kept free of methylation thereby facilitating the establishment of an open chromatin structure and of initiation of transcription. Besides its role in the regulation of genes, DNA methylation silences repetitive elements and appears to be important for the stability of the mammalian genome.
DNA-associated histone proteins play an important role in gene regulation within the mammalian genome. Various covalent chemical modifications of the histones are possible and can directly affect various DNA-templated processes such as transcription.
Polycomb and Trithorax group proteins are important epigenetic regulators of homeotic genes. They play a broad role in cell differentiation, which is exerted through the direct control of hundreds of transcription factors as well as important signaling proteins and morphogens. Polycomb silencing is a dynamic process intimately dependent on histone modifications and balanced by antagonistic action of Trithorax proteins.
In eukaryotic cells, the majority of transcribed RNAs are non-coding RNAs (ncRNAs). It is evident that ncRNAs are functionally involved in many biological processes, such as proliferation, differentiation and development. NcRNAs function as regulators of gene expression on various levels, including chromatin modification, transcription, RNA modification, RNA splicing, RNA stability and translation.
Genetic and epigenetic mechanisms contribute to the development of human tumors. Research into the differences between normal and cancer epigenomes, and the knowledge gained from single gene and large-scale epigenome analyses provide important information relevant to for gene discovery, therapeutic applications, and building a mechanism-based model of human tumorigenesis.
DNA methylation, chromatin structure, Polycomb proteins and non-coding RNAs are areas of current research and part of the fascinating and fast moving field of epigenetics.
Further reading: Epigenetics
Labels: epigenetics
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