Real-time PCR (original) (raw)
Real Time PCR; Applications in Diagnostics and Research
In recent years, real-time PCR has come forward as a robust and widely used molecular technique in clinical and biological settings. Although it can detect very minute quantities of target nucleic acid, but quantification of specific nucleic acids is not an easy task. Accurate and precise quantification is hampered by a number of factors that may include assay development and validation, fluorophores selection, handling during sample preparation, storage, reaction procedures, and batch analysis conditions. Even minor variations are significantly magnified by the exponential nature of this technique. Current review gives an insight of the advantages, limitations, assay chemistries, quantitation parameters, and quality control issues related to this technology . Moreover it will also highlight the utilization of Real time PCR in clinical oncology , virology , microbiology, and gene expression studies
PCR- Applications and Protocols
When approached about the possibility of editing another PCR volume, I thought the timing was appropriate given the explosion of PCR applications for mRNA quantitation, diagnosis, gene discovery, genomic analysis, and expression profiling. Also, although the newest crop of molecular biologists grew up using PCR routinely, I still find myself devoting significant time teaching first principles of PCR to help young investigators troubleshoot specific applications and to guide them through a confusing maze of choices as to which enzyme, buffer, cycling condition, etc., to use for which purpose.
Specificity, efficiency, and fidelity of PCR
Genome Research, 1993
The efficacy of PCR is measured by its specificity, efficiency (i.e. yield), and fidelity. A highly specific PCR will generate one and only one amplification product that is the intended target sequence. More efficient amplification will generate more products with fewer cycles. A highly accurate (i.e., high-fidelity) PCR, will contain a negligible amount of DNA polymerase-induced errors in its product. An ideal PCR would be the one with high specificity, yield, and fidelity. Studies indicate that each of these three parameters is influenced by numerous components of PCR, including the buffer conditions, the PCR cycling regime (i.e., temperature and duration of each step), and DNA polymerases. Unfortunately, adjusting conditions for maximum specificity may not be compatible with high yield; likewise, optimizing for the fidelity of PCR may result in reduced efficiency. Thus, when setting up a PCR, one should know which of the three parameters is the most important for its intended application and optimize PCR accordingly. For instance, for direct sequencing analysis of a homogenous population of ceils (either by sequencing or by RFLP), the yield and specificity of PCR is more important than the fidelity. On the other hand, for studies of individual DNA molecules, or rare mutants in a heterogeneous population, fidelity of PCR is vital. The purpose of current communication is to focus on the essential components of setting up an effective PCR, and discuss how each of these component may influence the specificity, efficiency, and fidelity of PCR. SETTING UP PCR Template Virtually all forms of DNA and RNA are suitable substrates for PCR. These include genomic (both eukaryotic and prokaryotic), plasmid, and phage DNA and previously amplified DNA, cDNA, and mRNA. Samples prepared via standard molecular methodologies (1) are sufficiently pure for PCR, and usually no extra purification steps are required. Shearing of genomic DNA during DNA extraction does not affect the efficiency of PCR (at least for the fragments that are less than-2 kb). In some cases, rare restriction enzyme digestion of genomic DNA before PCR is suggested to increase the yield. (2'3) In general, the efficiency of PCR is greater for smaller size template DNA (i.e., previously amplified fragment, plasmid, or phage DNA), than high molecular (i.e., undigested eukaryotic genomic) DNA. Typically, 0.1-1 pLg of mammalian genomic DNA is utilized per P e R. (1'3'4-6) Assuming that a haploid mammalian genome (3x109 bp) weighs-3. 4 x 10-az grams, 1 ~g of genomic DNA corresponds to-3 x 10 s copies of autosomal genes. For bacterial genomic DNA or a plasmid DNA that represent much less complex genome, picogram (10-12 grams) to nanogram (10-9 grams) quantities are used per reaction. (1'3) Previously amplified DNA fragments have also been utilized as PCR templates. In general, gel purification of the amplified fragment is recommended before the second round of PCR. Purification of the amplified product is highly recommended if the initial PCR generated a number of unspecific bands or if a different set of primers (i.e., internal primers) is to be utilized for the subsequent PCR. On the other hand, if the amplification reaction contains only the intended target product, and the purpose of the subsequent PCR is simply to increase the overall yield utilizing the same set of primers, no further purification is required. One could simply take out a small aliquot of the original PCR mixture and subject it to a second round of PCR. In addition to the purified form of DNA, PCR from cells has also been demonstrated. In this laboratory, direct amplification of hprt exon 3 fragment from 1 • 10 s human cells (following proteinase treatment to open up the cells) had been carried out routinely (P. Keohavong, unpubl.).
Review on Principles of Real Time Polymerase Chain Reaction (PCR
Review on Principles of Real Time Polymerase Chain Reaction (PCR), 2019
Real-time Polymerase chain reaction (Quantitative PCR), which allows for quantitative measurement of DNA or RNA molecules, is one among different kinds of PCR. Real-time PCR instrumentation was first made commercially available by Applied Bio-systems in 1996, after which several other companies added new machines to the market. The primary difference between real-time and conventional PCR assays is that products of a real time reaction are measured in "real time", as the PCR reaction is being performed, rather than after the reaction is complete. The basic goal of real-time PCR is to precisely distinguish and measure specific nucleic acid sequences in a sample even if there is only a very small quantity. Real-time PCR amplifies a specific target sequence in a sample then, monitors the amplification progress using fluorescent technology. A single molecular beacon is used for detection of a PCR amplification product and multiple beacon probes with different reporter dyes are used for single nucleotide polymorphism detection. The most commonly used methods for DNA preparation are based upon one of four systems: biphasic purification, silica-gel based column purification, magnetic bead purification and boiling with chelation of PCR inhibitors. Real-time PCR is used for absolute and relative quantifications of DNA and RNA template molecules and for genotyping in a variety of applications. As a limitation of Real time PCR, false-positive results are inevitable due to its high sensitivity, typically requiring no more than 10 copies of RNA or DNA for detection. When assays are adopted from other sources, careful attention should be paid to any variables which may not be identical; examples include changing the platform, using a different formulation of master mix, beginning with a different sample type or extraction method and changing either the template or reaction volume.
The real-time PCR methodology improved profoundly the basic amplification technology; not only by permitting the digital tracking of the reaction from the very first minutes, but also by significantly compressing the time for an amplification program, through technical improvements in expendables and hardware. Two of the integrated, end-to-end solutions available in the market, the Roche and the Applied Biosystems products, also capitalize on the advances at the multi-color dyes and the melting-curve analysis to perform products discrimination and dispose of post-amplification steps, such as electrophoresis, thus further compressing time and logistics footprint of the integrated assay. They also focus on the well-proven ability of the methodology to provide accurately quantitative results, which was always a priority throughout the field of biosciences, and one rarely tackled efficiently. Electrophoresis, though, remains popular with low-end users, who find it robust, reliable, flexible and relatively cheap since expendables are low-cost and know-how is well-established. Given that high-end users prefer true high-throughput methods (such as arrays) for simultaneous assaying, which vastly outperform real-time PCR, the ease and rapidity of the latter's results, which both are of capital diagnostic concern, remain the strongest points of the method, coupled with its quantification potential.
Applications Guide Real-Time PCR Applications Guide
In conventional PCR, the amplified product, or amplicon, is detected by an end-point analysis, by running DNA on an agarose gel after the reaction has finished. In contrast, real-time PCR allows the accumulation of amplified product to be detected and measured as the reaction progresses, that is, in "real time".
Polymerase Chain Reaction (PCR): Back to Basics
Advanced molecular technology has become a crucial tool for identifying new genes with importance in medicine, agriculture, animal production, health, environment, industry other related areas. Among the applications of molecular techniques is important to highlight the use of the Polymerase Chain Reaction (PCR) in the identification and characterization of viral, bacterial, parasitic and fungal agents. PCR is a process used in molecular biology to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Mechanisms involved in this methodology are similar to those occurring in vivo during DNA replication. Through this paper we will review procedure, advantages, types & applications of PCR.
Overview and recommendations for the application of digital PCR
2019
This publication is a Technical report by the Joint Research Centre (JRC), the European Commission's science and knowledge service. It aims to provide evidence-based scientific support to the European policymaking process. The scientific output expressed does not imply a policy position of the European Commission. Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use that might be made of this publication.
RT-PCR technique and its applications. State-of the-art
Journal of Animal and Feed Sciences, 2003
The reverse transcription polymerase chain reaction (RT-PCR) is one of the most sensitive methods for the detection and quantitation of mRNA. It is widely used for quantification of mRNA levels and is useful tool for basic research, agriculture, medicine and biotechnology. Introduction of Real-time technique significantly improves rapidity, sensitivity, specificity and reproducibility of RT-PCR method. Real-time PCR detects and quantifies nucleic acids even from live and dead pathogens or cells. This review discusses the background, advantages and limitations of conventional and Realtime RT-PCR methods for quantitation of gene expression and its application in basic, agricultural and biomedical research.