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.     

Synthesis and DNA binding studies of Ni(II), Co(II), Cu(II) and Zn(II) metal complexes of N1,N5-bis[pyridine-2-methylene]-thiocarbohydrazone Schiff-base ligand

The thiocarbohydrazone Schiff-base ligand with a nitrogen and sulphur donor was synthesized through condensation of pyridine-2-carbaldehyde and thiocarbohydrazide. Schiff-base ligands have the ability to conjugate with metal salts. A series of metal complexes with a general formula [MCl2(H2L)]·nH2O (MNi, Co, Cu and Zn) were synthesized by forming complexes of the N1,N5-bis[pyridine-2-methylene]-thiocarbohydrazone (H2L) Schiff-base ligand. These metal complexes and ligand were characterized by using ultraviolet-visible (UV-Vis), Fourier Transform Infrared (FT-IR), 1H and 13C NMR spectroscopy and mass spectroscopy, physicochemical characterization, CHNS and conductivity. The biological activity of the synthesized ligand was investigated by using Escherichia coli DNA as target. The DNA interaction of the synthesized ligand and complexes on E. coli plasmid DNA was investigated in the aqueous medium by UV-Vis spectroscopy and the binding constant (Kb) was calculated. The DNA binding studies showed that the metal complexes had an improved interaction due to trans-geometrical isomers of the complexes than ligand isomers in cis-positions.     

Banana pulp extract mediated synthesis of Cu2O nanoparticles: An efficient heterogeneous catalyst for the ipso-hydroxylation of arylboronic acids

A green and facile novel procedure has been developed for the synthesis of Cu₂O nanoparticles within a very short reaction time using banana pulp extract as a reducing agent. The synthesized nanoparticles are well characterized by SEM (Scanning Electron Microscope), TEM (Transmission Electron Microscope) and powder XRD (X-ray Diffraction) methods. An environmental benign and highly efficient protocol for the ipso-hydroxylation of aryl and hetero arylboronic acids using bio-fabricated Cu₂O nanoparticles as a catalyst and aqueous H₂O₂ as an oxidant has also been developed. The main advantages of this protocol are the base free reaction condition, reusable and heterogeneous catalytic system, and short reaction time with excellent yields.     

Synthesis of crescent shaped heterocycle-fused aromatics via Garratt-Braverman cyclization and their DNA-binding studies

Three crescent shaped heterocycle-fused phenanthrene based systems 1–3 have been synthesized starting from benzene (or substituted benzene) 1,2-bis-propargyl alcohols. Bis-alkylation with propargylic bromides provided the key intermediate, the bis-propargyl bis-ethers. In spite of the possibility of many competing reactions, the latter underwent facile double Garratt-Braverman cyclization to provide compounds 1–3 in near quantitative yield, in a striking reaction involving the formation of four C–C bonds in a single step. Compounds 1–3 showed binding interaction with DNA, predominantly, via groove binding along with partial intercalation (combilexins). Molecular docking study supported the proposed binding modes.     

First structural characterization of core modified 10,15-meso aryl azuliporphyrins: observation of C-H...pi interaction between pyrrole beta-CH and mesityl ring

First successful syntheses and structural characterization of new core modified meso aryl azuliporphyrins by a simple [3 + 1] methodology are reported.     

Core modified oxybenziporphyrins: new aromatic ligands for metal-carbon bond activation

Successful syntheses of two new aromatic core modified oxybenziporphyrins by a simple '3 + 1' methodology and the first aromatic core modified oxybenziporphyrin palladium complex are reported.     

Anion‐Directed Copper(II) Metallocages,Coordination Chain,and Complex Double Salt: Structures,Magnetic Properties,EPR Spectra,and Density Functional Study

A series of Cu^II metallo‐assemblies showing anion‐directed structural variations, including five metallocages [(G^n?)?{Cu₂(Hdpma)₄}]^(8?n)+(A^?)_8?n (G^n?=NO₃^?, ClO₄^?, SiF₆^2?, BF₄^?, SO₄^2?; A^?=NO₃^?, ClO₄^?, BF₄^?, CH₃SO₄^?; Hdpma=bis(3‐pyridylmethyl)ammonium cation), a complex double salt, namely, (H₃dpma)₄(CuCl₄)₅Cl₂, and a coordination chain, namely, [Cu₂(dpma)(OAc)₄], are reported. The influence of the anion can be explained by its coordinating ability, the affinity of which for the Cu^II center interferes significantly with metallocage formation, and its shape, which offers host–guest recognition ability to engage in weak metal–anion coordination and hydrogen bonding to the organic ligand, which are responsible for metallocage templation. EPR studies of these metallocages in the powder phase at room temperature and 77 K showed a trend of the g values (g_||>2.10>g_⊥>2.00) indicating a ‐based ground state with square‐pyramidal geometry for the Cu^II centers. The magnetism of these metallocages can be interpreted as the result of a combination of relatively small magnetic coupling integrals and a substantial contribution of temperature‐independent paramagnetism (TIP). The weak magnetic interaction is corroborated by the results of DFT calculations and the EPR spectra. Availability of the low‐lying state for spin population was confirmed by a magnetization study, which revealed a magnetic moment approaching 2Nβ, which would explain the presence of the larger TIP term.     

Surfactant-Assisted Acid Pretreatment of Sugarcane Tops for Bioethanol Production

Sugarcane tops is one of the largest biomass resources in India and in tropical countries such as Brazil in terms of surplus availability. Conversion of this feedstock to ethanol requires pretreatment to make it more accessible for the enzymes used in saccharification. Though several pretreatment regimens have been developed for addressing biomass recalcitrance, very few seem to be promising as an industrial process. A novel hybrid method involving use of mild acid and surfactant was developed which could effectively remove lignin and improve the sugar yield from sugar cane tops. Operational parameters that affect the pretreatment efficiency (measured as yield of sugars) were studied and optimized. Changes in structural properties of the biomass were studied in relation to the pretreatment process using scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier Transform Infrared (FTIR) analysis, and the changes in chemical composition was also monitored. The biomass pretreated with the optimized novel method could yield 0.798?g of reducing sugars per gram of pretreated biomass upon enzymatic hydrolysis.