SNP identification in unamplified human genomic DNA with gold nanoparticle probes

Single nucleotide polymorphisms (SNPs) comprise the most abundant source of genetic variation in the human genome. SNPs may be linked to genetic predispositions, frank disorders or adverse drug responses, or they may serve as genetic markers in linkage disequilibrium analysis. Thus far, established SNP detection techniques have utilized enzymes to meet the sensitivity and specificity requirements needed to overcome the high complexity of the human genome. Herein, we present for the first time a microarray-based method that allows multiplex SNP genotyping in total human genomic DNA without the need for target amplification or complexity reduction. This direct SNP genotyping methodology requires no enzymes and relies on the high sensitivity of the gold nanoparticle probes. Specificity is derived from two sequential oligonucleotide hybridizations to the target by allele-specific surface-immobilized capture probes and gene-specific oligonucleotide-functionalized gold nanoparticle probes. Reproducible multiplex SNP detection is demonstrated with unamplified human genomic DNA samples representing all possible genotypes for three genes involved in thrombotic disorders. The assay format is simple, rapid and robust pointing to its suitability for multiplex SNP profiling at the ‘point of care’

Multiplexed, rapid detection of H5N1 using a PCR-free nanoparticle-based genomic microarray assay

<p>Abstract</p> <p>Background</p> <p>For more than a decade there has been increasing interest in the use of nanotechnology and microarray platforms for diagnostic applications. In this report, we describe a rapid and simple gold nanoparticle (NP)-based genomic microarray assay for specific identification of avian influenza virus H5N1 and its discrimination from other major influenza A virus strains (H1N1, H3N2).</p> <p>Results</p> <p>Capture and intermediate oligonucleotides were designed based on the consensus sequences of the matrix (M) gene of H1N1, H3N2 and H5N1 viruses, and sequences specific for the hemaglutinin (HA) and neuraminidase (NA) genes of the H5N1 virus. Viral RNA was detected within 2.5 hours using capture-target-intermediate oligonucleotide hybridization and gold NP-mediated silver staining in the absence of RNA fragmentation, target amplification, and enzymatic reactions. The lower limit of detection (LOD) of the assay was less than 100 fM for purified PCR fragments and 10³TCID₅₀units for H5N1 viral RNA.</p> <p>Conclusions</p> <p>The NP-based microarray assay was able to detect and distinguish H5N1 sequences from those of major influenza A viruses (H1N1, H3N2). The new method described here may be useful for simultaneous detection and subtyping of major influenza A viruses.</p

Light-Activated Metal-Coordinated Supramolecular Complexes with Charge-Directed Self-Assembly

This document is the Accepted Manuscript version of a Published Work that appeared in final form in The Journal of Physical Chemistry C, copyright © American Chemical Society after peer review and technical editing by publisher. To access the final edited and published work see http://dx.doi.org/10.1021/jp3121403Metal-coordinated materials are attractive for many applications including catalysis, sensing, and controlled release. Adenine and its derivatives have been widely used to generate many coordination complexes, polymers, and nanoparticles. However, few of these materials display fluorescence. Herein, we report fluorescent gold complexes and nanoclusters formed with adenosine, deoxyadenosine, AMP, and ATP, where the former two produced micrometer-sized particles and the latter two produced molecular clusters. Only weak fluorescence was produced with adenine, while no emission was observed with uridine, cytidine, or guanosine. We found that adding citrate and light exposure are two key factors to generate fluorescence, and their mechanistic roles have been explored. In all the products, the ratio between gold and adenine was determined to be 1:1 using UV–vis spectroscopy. Mass spectrometry showed clusters containing 2, 4, and 6 gold atoms in the gas phase. The fluorescence peak is around 470 nm for the AMP and ATP complex and 480 nm for the (deoxy)adenosine complexes. This work has provided a systematic approach to obtain fluorescent metal coordinated polymers and materials with tunable sizes, which will find applications in analytical chemistry, drug delivery, and imaging. The fundamental physical chemistry of these materials has been systematically explored.University of Waterloo || Canadian Foundation for Innovation || Ontario Ministry of Research & Innovation || Natural Sciences and Engineering Research Council |

Analytical validation of the PAM50-based Prosigna Breast Cancer Prognostic Gene Signature Assay and nCounter Analysis System using formalin-fixed paraffin-embedded breast tumor specimens

Background: NanoString’s Prosigna™ Breast Cancer Prognostic Gene Signature Assay is based on the PAM50 gene expression signature. The test outputs a risk of recurrence (ROR) score, risk category, and intrinsic subtype (Luminal A/B, HER2-enriched, Basal-like). The studies described here were designed to validate the analytical performance of the test on the nCounter Analysis System across multiple laboratories. Methods: Analytical precision was measured by testing five breast tumor RNA samples across 3 sites. Reproducibility was measured by testing replicate tissue sections from 43 FFPE breast tumor blocks across 3 sites following independent pathology review at each site. The RNA input range was validated by comparing assay results at the extremes of the specified range to the nominal RNA input level. Interference was evaluated by including non-tumor tissue into the test. Results: The measured standard deviation (SD) was less than 1 ROR unit within the analytical precision study and the measured total SD was 2.9 ROR units within the reproducibility study. The ROR scores for RNA inputs at the extremes of the range were the same as those at the nominal input level. Assay results were stable in the presence of moderate amounts of surrounding non-tumor tissue (<70% by area). Conclusions: The analytical performance of NanoString’s Prosigna assay has been validated using FFPE breast tumor specimens across multiple clinical testing laboratories.Non UBCPathology and Laboratory Medicine, Department ofMedicine, Faculty ofReviewedFacult

Monoindenyl Titanium Alkyl Halides. The Synthesis and Molecular structures of (h\u3csup\u3e5\u3c/sup\u3e-C\u3csub\u3e9\u3c/sub\u3eH\u3csub\u3e7\u3c/sub\u3e)TiBr\u3csub\u3e3\u3c/sub\u3e, (h\u3csup\u3e5\u3c/sup\u3e-C\u3csub\u3e9\u3c/sub\u3eH\u3csub\u3e7\u3c/sub\u3e)Ti(CH\u3csub\u3e3\u3c/sub\u3e)Br\u3csub\u3e2\u3c/sub\u3e, and (h\u3csup\u3e5\u3c/sup\u3e-C\u3csub\u3e9\u3c/sub\u3eH\u3csub\u3e7\u3c/sub\u3e)Ti(CH\u3csub\u3e3\u3c/sub\u3e)Cl\u3csub\u3e2\u3c/sub\u3e

The monoindenyl titanium tribromide (η5-C9H7)TiBr3 (1), has been prepared from the interaction of TiBr4 and Bu3Sn&z.sbnd;C9H7. The monomethyl species (η5-C9H7)Ti(CH3)Br2 (2), and (η5-C9H7)Ti(CH3)Cl2 (3), have been efficiently prepared from the interaction of the trihalides with AlMe3. Complexes 1 and 2 represent rare examples of organotitanium bromides, and all three compounds are likely to be of interest in the context of homogeneous olefin polymerization catalysis. All three complexes have been characterized by 1H and 13C NMR spectroscopy and single crystal X-ray crystallography. These analyses reveal that the indenyl complexes exhibit a three-legged piano stool geometry in which the indenyl ligands are bound to the metal centers in an η5fashion. Crystal data for 1: (T=103 K), molecular formula, C9H7TiBr3, crystal system: monoclinic, space group P21/n, with a=7.155(1), b=12.682(1),c=12.216(1) Å, β=97.92°, V=1097.97 Å3, Z=4; 2: (T=103 K), molecular formula, C10H10TiBr2, crystal system: triclinic, space group P1, with a=7.142(1),b=12.501(1), c=7.117(1) Å, α=105.70(1), β=115.81(1), γ=88.21(1)°, V=547.91 Å3, Z=2; 3: (T=110 K), molecular formula, C10H10TiCl2, crystal system: orthorhombic, space group Pcab, with a=11.539(3), b=12.593(3), c=14.327(4) Å, V=2081.84 Å3, Z=8