Wisconsin Alumni Research Foundation

California Institute of Technology

Other

Third Wave Technologies, Inc.

Applied Biosystems, LLC

Northwestern University

Invitrogen Corporation

University of Illinois

Molecular Biology Resources, Inc.

Chimer

Lloyd M Smith

Michael S Westphall

Victor I Lyamichev

Bruce P Neri

Jeff G Hall

Steven Fung

Joshua J Coon

Mark Andrew Scalf

Brian L Frey

Michael Robert Shortreed

Leroy E Hood

Michael W Hunkapiller

Robert John Hamers

Sam L Woo

Robert H Blick

Charles R Connell

Todd C Strother

Robert J Kaiser, Jr

Cheng-Hsien Wu

Tim J Hunkapiller

Casey J Krusemark

Madhusudan Patel

Corinna Herrnstadt

David M Kelso

Joseph M Fernandez

Robert W Kwiatkowski, Jr

Peter Jeremy Belshaw

Hua Qin

Priscilla Wilkins Stevens

David A Mead

Robert E Webb

Ryan Tyler Hilger

Neil L Kelleher

William Travis Berggren

Robert L Brumley, Jr

Daniel D Ebeling

Margaret F Phillips

Shane M Lamos

Jason Dale Russell

Daniel T Ladror

Robert M Corn

Zhen Guo

Timothy Hunkapiller

Stephen C Weibel

Richard A Guilfoyle

Wei Cai

Xiaoyu Chen

April L Jue

Matthew R Lockett

Steven Fund

Anthony J Kostichka

Renfeng Wang

Bin Sun

Xinchun E Tong

Huamin Ji

H Blick Robert

The present invention is directed to methods and materials for RNA-mediated gene assembly from oligonucleotide sequences. In some embodiments, the oligonucleotides used for gene assembly are provided in an array format. An RNA polymerase promoter is appended to surface-bound oligonucleotides and a plurality of RNA copies of each oligonucleotide are then produced with an RNA polymerase. These RNA molecules self-assemble into a desired full-length RNA transcript by hybridization and ligation. The resulting RNA transcript may then be converted into double stand DNA useful in a variety of applications including protein expression.

RNA-mediated gene assembly from DNA oligonucleotides

Described herein are RNA arrays, and compositions and methods for generating RNA arrays, particularly high density RNA arrays. The disclosed methods for generating RNA arrays utilize template DNA arrays and RNA polymerase to generate RNA arrays. In some embodiments, the disclosed methods use an RNA polymerase and modified ribonucleosides to generate modified RNA arrays for various applications, e.g. RNA arrays having higher nuclease resistance, more conformationally stable RNA arrays, and higher binding affinity RNA aptamer arrays. In some embodiments, the disclosed methods are used to generate RNA bead arrays.

RNA array compositions and methods

The present invention is directed to methods and materials for RNA-mediated gene assembly from oligonucleotide sequences. In some embodiments, the oligonucleotides used for gene assembly are provided in an array format. An RNA polymerase promoter is appended to surface-bound oligonucleotides and a plurality of RNA copies of each oligonucleotide are then produced with an RNA polymerase. These RNA molecules self-assemble into a desired full-length RNA transcript by hybridization and ligation. The resulting RNA transcript may then be converted into double strand DNA useful in a variety of applications including protein expression.

SPR-compatible substrates for high density microarray fabrication and analyses are provided. Novel carbon-on-metal thin film substrate architecture permits the integration of surface plasmon resonance detection with photolithographically fabricated biomolecule arrays for the analysis of biomolecular interactions. The utility of the technology is shown in the analysis of specific DNA-DNA, DNA-RNA and DNA-protein binding interactions. These new substrates may be used to determine the secondary structure of RNA molecules, to probe the sequence-specific binding kinetics and affinity of proteins and small molecules, and as substrates for small-molecule combinatorial chemistry platforms for drug discovery applications.

Surface plasmon resonance compatible carbon thin films

An integrated mass spectrometer electrospray emitter and fluorescence detector allows improved volumetric measurements of separate components from a liquid chromatography column by improving correlation between the readings of these instruments and reducing dead volume and sample size requirements.

Integrated electrospray ionization emitter and detection cell for parallel measurements by fluorescence and mass spectrometry

An active detector and methods for detecting molecules, including large molecules such as proteins and oligonucleotides, at or near room temperature based on the generation of electrons via field emission (FE) and/or secondary electron emission (SEE). The detector comprises a semiconductor membrane having an external surface that is contacted by one or more molecules, and an internal surface having a thin metallic layer or other type of electron emitting layer. The kinetic energy of molecules contacting the semiconductor membrane is transferred through the membrane and induces the emission of electrons from the emitting layer. An electron detector, which optionally includes means for electron amplification, is positioned to detect the emitted electrons.

Molecule mass detection via field emission of electrons from membranes

A fast and sensitive method and device for protein sequencing are disclosed. The method uses a combination of Edman degradation chemistry and mass spectrometry to sequence proteins and polypeptides. A peptide degradation reaction is performed on a polypeptide or protein ion reactant in the gas phase. The reaction yields a first ion product corresponding to a first amino acid residue of the polypeptide or protein reactant and a polypeptide or protein fragment ion. The mass-to-charge ratio for the first ion product, or the polypeptide or protein fragment ion, or both, is then determined. The first amino acid residue of the polypeptide or protein reactant is then identified from the mass-to-charge ratio so determined.

Protein/peptide sequencing by chemical degradation in the gas phase

The present invention provides methods for enhancing the fragmentation of peptides for mass spectrometry by modifying the peptides with a tagging reagent containing a functional group, such as a tertiary amine, having a greater gas-phase basicity than the amide backbone of the peptide. These high gas-phase basicity functional groups are attached to a peptide by reacting the tagging reagent to one or more available carboxylic acid groups of the peptide. Linking these high gas-phase functional groups to the peptides leads to higher charge state ions from electrospray ionization mass spectrometry (ESI-MS), which fragment more extensively during fragmentation techniques, particularly non-ergodic fragmentation techniques such as electron capture dissociation (ECD) and electron transfer dissociation (ETD).

Labeling peptides with tertiary amines and other basic functional groups for improved mass spectrometric analysis

Relative quantification of metabolites by Electrospray Ionization Mass Spectrometry (ESI-MS) requiring a mechanism for simultaneous analysis of multiple analytes in two or more samples. Labeling reagents that are reactive to particular compound classes and differ only in their isotopic compositions facilitate relative quantification. Heavy and light isotopic forms of methylacetimidate were synthesized and used as labeling reagents for quantification of amine-containing molecules. Heavy and light isotopic forms of formaldehyde and cholamine were also synthesized and used independently as labeling reagents for quantification of amine-containing and carboxylic acid-containing molecules, such as found in biological samples. The labeled end-products are positively charged under normal acidic conditions involving conventional Liquid Chromatography Mass Spectrometry (LC/MS) applications. Labeled primary and secondary amine and carboxylic acid end-products generated higher signals concerning mass-spectra than pre-cursor molecules and improved sensitivity. Improved accuracy concerning relative quantification was demonstrated by mixing heavy and light labeledextracts in different ratios.

Ionizable isotopic labeling reagents for relative quantification by mass spectrometry

This invention provides a duplex comprising an oligonucleotide primer and a template, wherein the primer is coupled chemically to a chromophore or fluorophore so as to allow chain extension by a polymerase. In one embodiment, the primer is extended by a polymerase to generate the complement of the template. In a further embodiment, the extended primer is separated from the template for use in a number of methods, including sequencing reactions. Methods of generating these compositions of matter are further provided.

Tagged extendable primers and extension products

Relative quantification of metabolites by Electrospray Ionization Mass Spectrometry (ESI-MS) requiring a mechanism for simultaneous analysis of multiple analytes in two or more samples. Labeling reagents that are reactive to particular compound classes and differ only in their isotopic kit facilitating relative quantification and providing tangible evidence for the existence of specific functional groups. Heavy and light isotopic forms of methylacetimidate were synthesized and used as labeling reagents for quantification of amine-containing molecules, such as biological samples. Heavy and light isotopic forms of formaldehyde and cholamine were also synthesized and used independently as labeling reagents for quantification of amine-containing and carboxylic acid-containing molecules, such as found in biological samples. Advantageously, the labeled end-products are positively charged under normal acidic conditions involving conventional Liquid Chromatography Mass Spectrometry (LC/MS) applications. Labeled primary and secondary amine and carboxylic acid end-products also generated higher signals concerning mass-spectra than pre-cursor molecules and improved sensitivity. Improved accuracy concerning relative quantification was achieved by mixing heavy and light labeledextracts in different ratios. Labeling strategy was further employed to ascertain differences in the amounts of amine-containing metabolites for two strains ofseeds.

The present invention provides systems, devices, device components and structures for modulating the intensity and/or energies of electrons, including a beam of incident electrons. In some embodiments, for example, the present invention provides nano-structured semiconductor membrane structures capable of generating secondary electron emission. Nano-structured semiconductor membranes of this aspect of the present invention include membranes having an array of nanopillar structures capable of providing electron emission for amplification, filtering and/or detection of incident radiation, for example secondary electron emission and/or field emission. Nano-structured semiconductor membranes of the present invention are useful as converters wherein interaction of incident primary electrons and nanopillars of the nanopillar array generates secondary emission. Nano-structured semiconductor membranes of this aspect of the present invention are also useful as directed charge amplifiers wherein secondary emission from a nanopillar array provides gain functionality for increasing the intensity of radiation comprising incident electrons.

Nanopillar arrays for electron emission

The present invention relates to compositions and methods for the detection and characterization of nucleic acid sequences and variations in nucleic acid sequences. The present invention relates to methods for forming a nucleic acid cleavage structure on a solid support and cleaving the nucleic acid cleavage structure in a site-specific manner. For example, in some embodiments, a 5' nuclease activity from any of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

Reactions on a solid surface

Chemically-modified surfaces on unoxidized carbon, silicon, and germanium substrates are disclosed. Ultraviolet radiation mediates the reaction of protected &#x3c9;-modified, &#x3b1;-unsaturated aminoalkenes (preferred) with hydrogen-terminated carbon, silicon, or germanium surfaces. Removal of the protecting group yields an aminoalkane-modified silicon surface These amino groups can be coupled to terminal-modified oligonucleotides using a bifunctional crosslinker, thereby permitting the preparation of modified surfaces and arrays. Methods for controlling the surface density of molecules attached to the substrate are also disclosed.

Modified carbon and germanium surfaces

Growing community of inventors

Madison, WI, United States of America

Lloyd M Smith