Article

The real-time polymerase chain reaction.

TATAA Biocenter, Medicinargatan 7B, 405 30 Göteborg, Sweden.
Molecular Aspects of Medicine (Impact Factor: 10.3). 04/2006; 27(2-3):95-125. DOI: 10.1016/j.mam.2005.12.007
Source: PubMed

ABSTRACT The scientific, medical, and diagnostic communities have been presented the most powerful tool for quantitative nucleic acids analysis: real-time PCR [Bustin, S.A., 2004. A-Z of Quantitative PCR. IUL Press, San Diego, CA]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies [Saiki, R.K., et al., 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350], for which Kary Mullis was awarded the 1993 year's Nobel prize in Chemistry. By PCR essentially any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. This made it possible, for example, to manipulate DNA for cloning purposes, genetic engineering, and sequencing. But as an analytical technique the original PCR method had some serious limitations. By first amplifying the DNA sequence and then analyzing the product, quantification was exceedingly difficult since the PCR gave rise to essentially the same amount of product independently of the initial amount of DNA template molecules that were present. This limitation was resolved in 1992 by the development of real-time PCR by Higuchi et al. [Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992. Simultaneous amplification and detection of specific DNA-sequences. Bio-Technology 10(4), 413-417]. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification efficiency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR.

0 Followers
 · 
249 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Invasive Asian bighead and silver carp (Hypophthalmichthys nobilis and H. molitrix) pose a substantial threat to North American aquatic ecosystems. Recently, environmental DNA (eDNA), genetic material shed by organisms into their environment that can be detected by non-invasive sampling strategies and genetic assays, has gained recognition as a tool for tracking the invasion front of these species toward the Great Lakes. The goal of this study was to develop new species-specific conventional PCR (cPCR) and quantitative (qPCR) markers for detection of these species in North American surface waters. We first generated complete mitochondrial genome sequences from 33 bighead and 29 silver carp individuals collected throughout their introduced range. These sequences were aligned with those from other common and closely related fish species from the Illinois River watershed to identify and design new species-specific markers for the detection of bighead and silver carp DNA in environmental water samples. We then tested these genetic markers in the laboratory for species-specificity and sensitivity. Newly developed markers performed well in field trials, did not have any false positive detections, and many markers had much higher detection rates and sensitivity compared to the markers currently used in eDNA surveillance programs. We also explored the use of multiple genetic markers to determine whether it would improve detection rates, results of which showed that using multiple highly sensitive markers should maximize detection rates in environmental samples. The new markers developed in this study greatly expand the number of species-specific genetic markers available to track the invasion front of bighead and silver carp and will improve the resolution of these assays. Additionally, the use of the qPCR markers developed in this study may reduce sample processing time and cost of eDNA monitoring for these species.
    PLoS ONE 02/2015; 10(2):e0117803. DOI:10.1371/journal.pone.0117803 · 3.53 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We have developed a label-free viscosity-based DNA detection system, using paramagnetic beads as an asynchronous magnetic bead rotation (AMBR) microviscometer. We have demonstrated experimentally that the bead rotation period is linearly proportional to the viscosity of a DNA solution surrounding the paramagnetic bead, as expected theoretically. Simple optical measurement of asynchronous microbead motion determines solution viscosity precisely in microscale volumes, thus allowing an estimate of DNA concentration or average fragment length. The response of the AMBR microviscometer yields reproducible measurement of DNA solutions, enzymatic digestion reactions, and PCR systems at template concentrations across a 5000-fold range. The results demonstrate the feasibility of viscosity-based DNA detection using AMBR in microscale aqueous volumes.
  • [Show abstract] [Hide abstract]
    ABSTRACT: We identified two lysozyme cDNA isoforms from starry flounder Platichthys stellate. The c-type and g-type starry flounder lysozyme cDNAs encoded polypeptides of 143 and 188 amino acids, respectively. Based on bioinformatic sequence characterizations, both isoforms possessed catalytic and other conserved residues essential for their functionality. Tissue distribution analysis revealed that c-type and g-type lysozyme transcripts were ubiquitously found in all tissue types examined. In particular, the c-type lysozyme mRNA was highly predominant in the spleen. On the other hand, g-type lysozyme transcripts were markedly detectable in posterior intestine, followed by kidney and spleen. In response to Streptococcus parauberis challenge, the splenic c-type, but not g-type, lysozyme mRNA level was significantly induced, indicating that the spleen is a major lysozyme producer in response to bacterial infection-mediated stress condition. At protein levels, the enhanced lysozyme activities in the skin mucus and serum were observed as early as 4 h post injection, suggesting that the lysozyme could be involved in the acute phase responses against bacterial infection. Taken together, our data indicate that S. parauberis infection elicits a lysozyme-associated systemic immune response in starry flounder, possibly in a tissue-specific and/or isoform-specific manner.
    Fisheries Science 03/2015; 81(2). DOI:10.1007/s12562-015-0852-0 · 0.86 Impact Factor

Full-text (2 Sources)

Download
91 Downloads
Available from
May 27, 2014