Multiplex digital PCR: breaking the one target per color barrier of quantitative PCR

Quantitative polymerase chain reactions (qPCR) based on real-time PCR constitute a powerful and sensitive method for the analysis of nucleic acids. However, in qPCR, the ability to multiplex targets using differently colored fluorescent probes is typically limited to 4-fold by the spectral overlap of the fluorophores. Furthermore, multiplexing qPCR assays requires expensive instrumentation and most often lengthy assay development cycles. Digital PCR (dPCR), which is based on the amplification of single target DNA molecules in many separate reactions, is an attractive alternative to qPCR. Here we report a novel and easy method for multiplexing dPCR in picolitre droplets within emulsions-generated and read out in microfluidic devices-that takes advantage of both the very high numbers of reactions possible within emulsions (>10(6)) as well as the high likelihood that the amplification of only a single target DNA molecule will initiate within each droplet. By varying the concentration of different fluorogenic probes of the same color, it is possible to identify the different probes on the basis of fluorescence intensity. Adding multiple colors increases the number of possible reactions geometrically, rather than linearly as with qPCR. Accurate and precise copy numbers of up to sixteen per cell were measured using a model system. A 5-plex assay for spinal muscular atrophy was demonstrated with just two fluorophores to simultaneously measure the copy number of two genes (SMN1 and SMN2) and to genotype a single nucleotide polymorphism (c.815A>G, SMN1). Results of a pilot study with SMA patients are presented.     

Quantitative and sensitive detection of rare mutations using droplet-based microfluidics

Somatic mutations within tumoral DNA can be used as highly specific biomarkers to distinguish cancer cells from their normal counterparts. These DNA biomarkers are potentially useful for the diagnosis, prognosis, treatment and follow-up of patients. In order to have the required sensitivity and specificity to detect rare tumoral DNA in stool, blood, lymph and other patient samples, a simple, sensitive and quantitative procedure to measure the ratio of mutant to wild-type genes is required. However, techniques such as dual probe TaqMan(?) assays and pyrosequencing, while quantitative, cannot detect less than ～1% mutant genes in a background of non-mutated DNA from normal cells. Here we describe a procedure allowing the highly sensitive detection of mutated DNA in a quantitative manner within complex mixtures of DNA. The method is based on using a droplet-based microfluidic system to perform digital PCR in millions of picolitre droplets. Genomic DNA (gDNA) is compartmentalized in droplets at a concentration of less than one genome equivalent per droplet together with two TaqMan(?) probes, one specific for the mutant and the other for the wild-type DNA, which generate green and red fluorescent signals, respectively. After thermocycling, the ratio of mutant to wild-type genes is determined by counting the ratio of green to red droplets. We demonstrate the accurate and sensitive quantification of mutated KRAS oncogene in gDNA. The technique enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines and the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of unmutated KRAS genes. The sensitivity is only limited by the number of droplets analyzed. Furthermore, by one-to-one fusion of drops containing gDNA with any one of seven different types of droplets, each containing a TaqMan(?) probe specific for a different KRAS mutation, or wild-type KRAS, and an optical code, it was possible to screen the six common mutations in KRAS codon 12 in parallel in a single experiment.     

Mixing enhancement in a multi-stream injection nozzle

We present quantitative analysis of image sequences of multi-stream injection nozzle flows with several different injection geometries in an experiment simulating mixing in a chemical oxygen-iodine laser. To visualize mixing, image sequences were acquired with planar laser-induced fluorescence (PLIF) in iodine that was injected into the main flow. The injection nozzle consisted of a slot, ejector, and injector block, with rows of ejector and injector holes along the slot length. The ejector flow exits in an underexpanded state so that upon expanding it forces the slot and injector flows together to enhance mixing. For this study, the diameter and geometry of ejector holes were varied to assess their effect on mixing. Two configurations of ejector holes were used, each with two different diameters for a total of four cases with data collected at downstream stations. We carry out a quantitative mixing analysis for these configurations, using two methods to quantify the mixing. The first method considers the statistics of the PLIF image intensity histograms, which are bimodal for poorly-mixed flows and have a single peak in well-mixed flows. The second method quantifies the properties of the mixing interface. Our analysis shows that two injection schemes significantly enhance mixing by stretching the mixing interface.     

The Assignment of the 1600 cm−1 Mystery Band of Carbons

Abstract

The assignment of a band near 1600 cm^?1 in IR spectra of carbons has been controversial for four decades. However, many different carbons have been studied: effectively, a single band assignment was sought for an absorption appearing with three different classes of carbon. As these differ in over-all structure, not one but three explanations are needed. These are discussed. However, undue emphasis has been placed on a single absorption; attention should also be paid to other absorptions accompanying the 1600 cm^?1 band.     

The Cesàro operator on the Bergman space $ A^{2} (\mathbb{D}) $

The Cesàro operator is shown to be subdecomposable on the Bergman spaces A^p (\mathbbD) A^{p} (\mathbb{D}) for p \geqq 2 p \geqq 2 , extending a result of [12] to the case that p < 4. For A² (\mathbbD) A^{2} (\mathbb{D}) , we show that Cesàro operator is in fact subscalar, but in contrast to the situation in the Hardy space, C |_A² C |_{A^2} fails to be hyponormal.     

Photochemistry in ordered physisorbed monolayers of dinitrogen tetroxide

The effects of physisorption and two-dimensional ordering on the photochemistry of N₂O₄ were investigated. Ordered monolayers were prepared by adsorption of NO₂ at 100 K on a water-ice surface. Irradiation with a continuous light source in the wavelength region 300–400 nm or with pulsed laser radiation at 355 nm resulted in exclusive desorption of NO₂. This desorption was induced by electronic absorption directly in the adsorbate via a transition corresponding to the ( )¹B_2u←( )¹A_g transition in N₂O₄, as in the gas phase. However, the subsequent dynamics in the excited state were markedly different from the gas-phase counterpart. Time-of-flight mass spectrometry of NO₂ photodesorbed at 355 nm revealed a most probable fragment translational energy of ca. 17 meV; and the angular distribution of the nascent NO₂ was peaked sharply in a direction around 10° from the normal. It is apparent that, despite the weak interaction with the substrate, significant energy transfer occurs in the ordered physisorbed monolayer to yield nascent NO₂ with very low translational energy and a constrained angle of escape which is consistent with a high degree of adsorbate order and alignment.     

Electrochemical reduction of pyrethroid insecticides based on esters of α-cyano-3-phenoxybenzyl alcohol at glassy carbon and mercury electrodes in acetonitrile

The electrochemical reduction of eight commercially important pyrethroid insecticides which are esters of either α-cyano-3-phenoxybenzyl alcohol (cycloprothrin, cyphenothrin, cyhalothrin, deltamethrin, esfenvalerate and cypermethrin) or 4-fluro-α-cyano-3-phenoxybenzyl alcohol (cyfluthrin and flumethrin) has been studied under conditions of voltammetry and bulk electrolysis at both glassy carbon and mercury electrodes in acetonitrile. In general, the peak potential of the initial reduction process observed at very negative potentials at both electrode surfaces shifts to a more positive value under conditions of consecutive potential cycling. At the hanging mercury drop electrode the reduction occurs at even more negative potentials than at a glassy carbon electrode because a blocking mechanism appears to be operative. Despite this major difference in the primary reduction step, common voltammetric features are observed at less negative potentials on second and subsequent cycles of the electrode potential at either electrode surface. For example, the initial reduction process always results in the formation of a species which is reversibly reduced at less negative applied potentials. Furthermore, despite the definition of the voltammetric response being highly sensitive to the individual pyrethroid structure, long time-scale bulk electrolysis experiments at glassy carbon or mercury pool electrodes led to the formation of analogous final products. The fact that pyrethroids with a widely varying range of acid moieties exhibit similar voltammetric behaviour suggests that the acid moiety is not directly involved in the initial electron transfer process. Controlled potential electrolysis studies at both electrode surfaces coupled with HPLC and mass spectral identification of products obtained after ethylation with ethyl iodide showed that the reduction mechanism on the longer time-scale involves cleavage of the ester with liberation of free cyanide ion. The major reduction product identified was the anion of either 3-phenoxybenzoic acid or 4-fluoro-3-phenoxybenzoic acid in yields ranging from 31 to 66%.