Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and ...real-time PCR revealed greater precision (coefficients of variation decreased 37-86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer.
Droplet digital polymerase chain reaction (ddPCR) is a new technology that was recently commercialized to enable the precise quantification of target nucleic acids in a sample. ddPCR measures ...absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. This novel ddPCR format offers a simple workflow capable of generating highly stable partitioning of DNA molecules. In this study, we assessed key performance parameters of the ddPCR system. A linear ddPCR response to DNA concentration was obtained from 0.16% through to 99.6% saturation in a 20,000 droplet assay corresponding to more than 4 orders of magnitude of target DNA copy number per ddPCR. Analysis of simplex and duplex assays targeting two distinct loci in the Lambda DNA genome using the ddPCR platform agreed, within their expanded uncertainties, with values obtained using a lower density microfluidic chamber based digital PCR (cdPCR). A relative expanded uncertainty under 5% was achieved for copy number concentration using ddPCR. This level of uncertainty is much lower than values typically observed for quantification of specific DNA target sequences using currently commercially available real-time and digital cdPCR technologies.
Characterizing the transcriptome of individual cells is fundamental to understanding complex biological systems. We describe a droplet-based system that enables 3' mRNA counting of tens of thousands ...of single cells per sample. Cell encapsulation, of up to 8 samples at a time, takes place in ∼6 min, with ∼50% cell capture efficiency. To demonstrate the system's technical performance, we collected transcriptome data from ∼250k single cells across 29 samples. We validated the sensitivity of the system and its ability to detect rare populations using cell lines and synthetic RNAs. We profiled 68k peripheral blood mononuclear cells to demonstrate the system's ability to characterize large immune populations. Finally, we used sequence variation in the transcriptome data to determine host and donor chimerism at single-cell resolution from bone marrow mononuclear cells isolated from transplant patients.
Haplotyping of human chromosomes is a prerequisite for cataloguing the full repertoire of genetic variation. We present a microfluidics-based, linked-read sequencing technology that can phase and ...haplotype germline and cancer genomes using nanograms of input DNA. This high-throughput platform prepares barcoded libraries for short-read sequencing and computationally reconstructs long-range haplotype and structural variant information. We generate haplotype blocks in a nuclear trio that are concordant with expected inheritance patterns and phase a set of structural variants. We also resolve the structure of the EML4-ALK gene fusion in the NCI-H2228 cancer cell line using phased exome sequencing. Finally, we assign genetic aberrations to specific megabase-scale haplotypes generated from whole-genome sequencing of a primary colorectal adenocarcinoma. This approach resolves haplotype information using up to 100 times less genomic DNA than some methods and enables the accurate detection of structural variants.
Mechanism of Permanganate Chemiluminescence Hindson, Christopher M; Francis, Paul S; Hanson, Graeme R ...
Analytical chemistry (Washington),
05/2010, Letnik:
82, Številka:
10
Journal Article
Recenzirano
Spectroscopic and synthetic methods have been exploited to deduce the mechanism for acidic potassium permanganate chemiluminescence. We have employed electron paramagnetic resonance (EPR) ...spectroscopy with a continuous flow assembly to monitor the formation of radical intermediates in real time generated from substrate oxidation by manganese(VII). These transient species react with manganese(III) in solution to produce the previously characterized manganese(II)* emission source. Using UV−vis, EPR, attenuated total reflection (ATR)-FT-IR, and chemiluminescence spectroscopies, we have established that there are two distinct enhancement mechanisms that in combination afford a 50-fold increase in emission intensity when the reaction is conducted in the presence of phosphate oligomers. In addition to preventing disproportionation of the manganese(III) precursor, the phosphate oligomers form protective “cagelike” structures around the manganese(II)* emitter, thus preventing nonradiative relaxation pathways.
Two years ago, we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue ...standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a “second generation” ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from nonspecific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.
Electron paramagnetic resonance spectroscopy has afforded the identification of a much postulated 1,4-dioxy biradical that occurs within the light producing pathway of peroxyoxalate chemiluminescence.
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► The rate of reaction and chemiluminescence intensity are highly analyte dependent. ► The selectivity of reagent can be tuned by manipulating reaction conditions. ► Acids and ...polyphosphates both stabilise the key Mn(III) precursor to the emitter. ► Formaldehyde and formic acid generate higher concentrations of Mn(III).
Examination of the chemiluminescence reactions of dihydroxybenzenes, aminophenols and phenylenediamines with acidic potassium permanganate has provided a new understanding of the relationships between analyte structure, reaction conditions, kinetics of the light-producing pathway and emission intensity, with broad implications for this widely utilised chemiluminescence detection system. Using a permanganate reagent prepared in a polyphosphate solution and adjusted to pH 2.5, large differences in the rate of reaction with different positional isomers were observed, with the
meta-substituted forms reacting far slower and therefore exhibiting much lower chemiluminescence intensities in flow analysis systems. The preliminary partial reduction of permanganate to form significant concentrations of Mn(III) increased the rate of reaction with all analytes tested, resulting in comparable or (in the case of aminophenol and phenylenediamine) even greater emission intensities for the
meta-isomers, demonstrating the opportunity to tune the selectivity of the reagent towards certain classes of compound or even specific positional isomers of the same compound. Using more acidic permanganate reagents, in which polyphosphates are not required, the discrepancy between the chemiluminescence intensities was still observed, but was less prominent due to the generally faster rates of reaction. The enhancement of these chemiluminescence reactions by on-line addition of formic acid or formaldehyde can in part also be attributed to the generation of significant pools of the key Mn(III) precursor to the emitting species.
Manganese(II) salts catalyze the chemiluminescent oxidation of organic compounds with acidic potassium permanganate. The formation of insoluble manganese(IV) species from the reaction between ...manganese(II) and permanganate can be prevented with sodium polyphosphate, and therefore, relatively high concentrations of the catalyst can be added to the reagent before the light-producing reaction is initiated. The rapid and intense emissions from these manganese(II) catalyzed chemiluminescence reactions provide highly sensitive detection and greater compatibility with liquid chromatography.
Enhanced permanganate chemiluminescence FRANCIS, Paul S; HINDSON, Christopher M; TERRY, Jessica M ...
Analyst (London),
01/2011, Letnik:
136, Številka:
1
Journal Article
Recenzirano
The significant enhancement of acidic potassium permanganate chemiluminescence by Mn(II) results from the concomitant presence of permanganate and Mn(III) in the reagent solution, which enables rapid ...production of the excited Mn(II) emitter with a wide range of analytes. Furthermore, the key Mn(III) co-reactant can be quickly generated by reducing permanganate with sodium thiosulfate, instead of the slow (~24 h) equilibration required when Mn(ii) is used. The emission from reactions with analytes such as tyrosine and fenoterol was over two orders of magnitude more intense than with the traditional permanganate reagent.
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