Research Tools : Genomics & proteomics


Nanopore Antennas for Ultrahigh Speed DNA Sequencing

Building on their work, the researchers have now developed metallic nanopores for ultrahigh speed molecule sequencing. The new nanopores are electrically conductive and function as antennas, transmitting radiofrequency signals with utmost precision.

Unlike competing technology, the nanopores feature both genetically and electrically engineered components. They can be constructed of DNA attached with metal particles to enhance electromagnetic wave reception. This is achieved by replacing the side chains of the DNA molecule with sulfur groups that in turn link to gold particles. Metalized DNA strands or ‘arms’ can be added to increase antenna size and tune polarization.

Identifying Related Peak Sets to Boost Mass Spectrometry Throughput

UW–Madison researchers have developed an algorithm for identifying related peak sets from MS1 spectra data.

First, an intensity peak is selected from the MS1 data and its peak location is identified. Based on intensity values associated with all potentially related peak locations, an intensity score is calculated. This score determines whether or not the peak locations form a related set. Related peaks may optionally be selected for MS2 processing.

DNA Sequencing with Piezoelectric Nanopore

UW–Madison researchers have developed a method for adjusting in situ the diameter of a nanopore used in a DNA sequencing device via piezoelectric tuning. The ability to control pore dimensions helps control the speed of the material passing through.

The substrate of the device is made of a piezoelectric material like quartz, which physically strains in response to an electric field. The substrate is positioned between reservoirs of conductive fluid and forms a nanoscale opening for DNA and ions. When an electrical signal is applied by a pair of electrodes on either side, the diameter of the opening changes due to piezoelectric shear strain. This constriction slows the passage of DNA through the pore long enough to identify one nucleotide at a time.

Cost-Effective Isobaric Tandem Mass Tags for High Throughput Quantitative Proteomics and Peptidomics

UW–Madison researchers have designed and synthesized novel N,N-dimethylated amino acid eight- and 16-plex isobaric MS/MS tagging reagents.

The reagents consist of a reporter group and a balancing group that are isotopically coded to provide eight compounds with equal molecular weights. The balancing group is designed to provide eight isotopic combinations. The reagents feature an amine reactive group capable of reacting with the molecule to be tagged. Compared to iTRAQ reagents, the eight-plex dimethyl leucine reagents also give rise to high intensity parent and reporter ions, offering enhanced sensitivity and dynamic range for detection and quantitation of low-abundance analytes.

GFAbs – GFP-Based Biosensors with the Binding Properties of Antibodies

UW-Madison researchers have developed a GFP-based scaffold that maintains its fluorescence properties in the presence of two inserted binding loops.  The scaffold is capable of accepting a diverse loop repertoire from which fluorescent binding proteins could be isolated.

Inserting multiple loops into the scaffold yields fluorescent biosensors known as GFAbs.  The researchers have developed expression libraries consisting of multiple fluorescent biosensors, which are capable of detecting and isolating antigens or other molecules of interest, to provide a resource for identifying binding ligands.

Increasing Peptide and Protein Identifications with Prioritized Mass Spectrometry

UW–Madison researchers have developed a universally compatible, computationally based method in which multiple precursor ion attributes—such as mass, intensity, m/z ratio and charge state, as well as results obtained from previous scans—are used to calculate the likelihood of identification, thus prioritizing subsequent analysis.

The method comprises MS/MS analysis of a sample (or training sample) containing proteins and peptides, with one or more compounds optionally labeled with isobaric tags. Established procedure for analyte ionization and mass-to-charge separation generates precursor ions. The ions then are detected and analyzed for information related to two or more physical properties (mass, charge state, etc.) and directed for MS2 dissociation—selecting and/or allotting resources to the most identifiable ions as determined by the algorithm. Segregated and detected for mass and abundance, the fragmented ions provide further characteristic data used to identify the compounds. Additionally, the process permits novel, or first-time, identification of precursor ions during an experiment or within an ID database.

Enabling prioritized, probability-focused mass spectrometry, the innovative software achieves increased sample identification with little or no time increase and requiring no additional hardware.

Mass Spectrometry Data Acquisition Method Enables More Reliable Large-Scale Protein Quantitation

UW–Madison researchers have developed a platform for analyte quantitation that prevents the acquisition of mass spectra that will not result in usable data due to interference by modifying the data acquisition software. During the automated precursor selection process, candidate precursors with significant interference are rejected until a suitable replacement is found. This method enables increased quantitation accuracy while maintaining high levels of throughput.

Specifically, a distribution of precursor ions from an analyte is analyzed using mass spectrometry to identify a precursor peak in the ion mass spectrometry data corresponding to a precursor ion. The data allows determination of the amount of interference within a preselected range about the precursor peak. Then, an adjusted range of ions may be selected for analysis such that the amount of interference is less than a selected value.

Double-Strand DNA Break Repair in Vitro – Forensic and Genomic Applications

UW–Madison researchers have developed a method for the in vitro joining of two DNA fragments that have homologous DNA sequences through a simplified process of DNA double-strand break repair.  This approach enables the sequencing of poor quality or minimally available DNA that may be in a complex mixture of contaminating DNA sequences.  It requires only three proteins or their homologues: RecA protein, single-stranded DNA binding protein (SSB) and DNA polymerase I.  The proteins preferably are obtained from E. coli

In this method, a single-stranded DNA probe with some homology to the target DNA is combined with RecA protein and SSB.  Then the target duplex DNA molecule, which has a double-strand break and is not super-coiled, is added to the mixture.  The single-strand probe invades the doubled-stranded target DNA.  When DNA polymerase is added with dNTPs, it extends both strands to create a double-stranded DNA molecule in which the two fragments have joined.  PCR reactions then can be carried out using primers that bind the probe and target DNA.  This will allow amplification of STRs, if there are lesions in the DNA close to an STR.

Use of Nanomaterials to Enrich Phosphopeptides for Mass Spectrometry-Based Proteomics

UW–Madison researchers have developed a method and materials for isolating, purifying and enriching the concentration of compounds containing phosphate groups, including phosphorylated peptides and proteins. The mesoporous nanomaterials are made from transition metal oxides, which selectively and reversibly bind phosphorylated compounds. These materials are relatively easy to prepare and have nanopore structures attractive for enrichment due to large surface area, high flow-through capacity, chemical stability and robustness.

In practice, the enrichment materials are contacted with a sample containing a mixture of phosphorylated and non-phosphorylated compounds. When contacted with the mesoporous enrichment material, the phosphorylated compounds reversibly bind to the surface while the remainder of the solution passes through the column. Once separated, the phosphorylated compounds are then removed from the mesoporous metal oxide enrichment material via controlled release.

These methods and materials are highly versatile and can be used for highly efficient enrichment, purification and effective analysis of phosphorylated compounds in a variety of biological environments. They also are highly complementary to both bottom-up and top-down mass spectrometric-based protein identification methods and can be used to effectively apply these methods in the study of proteomics.

Real-Time Tandem Mass Spectral Data Analysis for Protein Sequence Identification

UW-Madison researchers have developed a technique that allows for real-time identification of unknown peptides using mass spectral analysis to identify and characterize proteins during a tandem mass spectral analysis. 

With sufficient MS resolution, the z-type product ions of the ETD technique can be uniquely identified, in turn allowing for immediate identification of c-type product ions.  Because of the labeling of both product ions, other spectra peaks due to noise can be eliminated and the overall spectra quality can be determined.  The masses of the z- and c-type product ions are compared against a computer database to identify one or more “putative chemical compositions,” which are the amino acid sequences of the peptide.  The putative chemical compositions then are confirmed by comparison to peptide amino acid sequences in a database or via de novo analysis using a computational model without extrinsic comparison.  The results from either of these methods can be used to identify and characterize the protein from which the peptide was derived.  This process is incorporated into an algorithm that can make automated decisions to determine the best course of action for the mass spectral analysis.

Efficient Whole Genome Analysis System

UW-Madison researchers have developed an efficient whole genome analysis system, called “Nanocoding,” which acquires sequence information from numerous large DNA molecules in new ways.  Because large molecules are used, this system obtains information from heterochromatic regions, pinpoints structural variants and can even characterize aberrations associated with cancer cell genomes.

In this system, a new class of restriction endonucleases that cleaves just one strand of DNA is used to introduce sequence-specific nick sites into large genomic DNA molecules.  After labeling, this action results in ordered restriction maps for the individual molecules.  The barcoded maps can be aligned with reference maps to identify each molecule and reveal structural alterations, such as missing restriction sites, insertions or deletions.

A key component of this approach is the termination of DNA strand synthesis at a known distance away from a given nick site that has been mapped.  This distance defines the breadth of a “neighborhood.”  Labeled nucleotides are incorporated into each neighborhood between the nick and termination sites.  The amount of label, which defines the measurable optical “signature” of the neighborhood, depends on the nucleotide sequence in the region.  The set of all signatures within a given DNA molecule makes up its “barcode,” which is used to compare local sequence composition against that of a reference genome.

Accurate Determination of Metabolite Concentrations in Cell Extracts or Biological Fluids

UW-Madison researchers have developed a simple experimental protocol and device for identifying and measuring concentrations of metabolites in complex solutions from 2-D 1H-13C NMR spectra. This protocol may lead to a faster, more consistent and accurate method for generating more complete metabolite concentration information.

The intensity peaks from the NMR data are used to identify metabolites in advance. Then a processor calculates the concentration of each metabolite compound, based on the concentration equation associated with the compound and the intensity value from the NMR data.

Synthetic Cofactor Analogs of S-Adenosylmethionine as Ligatable Probes of Biological Methylation

UW-Madison researchers have developed compounds and methods for specifically labeling the substrates of SAM-dependent methyltransferases. The methods use SAM analogs that have been modified at the C5` position so the analog is transferred by the methyltransferase to a methylation site in a substrate, such as a peptide or nucleic acid. Once anchored to the substrate, these cofactor analogs allow for the addition of a detectable and/or isolable label. The label may contain various moieties that aid in determining the methylation state of the substrate. The SAM analogs can also be used with nucleic acid methyltransferases to allow for the rapid identification of specific DNA or RNA residues that are typically methylated.

Electrospray Ionization Ion Source with Tunable Charge Reduction

The researchers have improved the physical layout of that device by moving the corona discharge and its associated electromagnetic fields outside the charge reduction chamber, making it easier to collect the ions in the mass spectrometer for analysis. This device configuration also provides independent control over the conditions and processes involved in analyte and reagent ion formation, avoids perturbations in the trajectories of analyte ions and charged droplets caused by operation of the reagent ion source, and allows use of a wide range of reagent ion sources.

Computer-Based Determination of Haplotype

UW–Madison researchers have developed a computer-based method for determining haplotypes. Their system uses genotype data obtained from a wide range of mapping techniques (e.g., experiments with single molecules, families or populations).

The new system identifies polymorphic markers (SNPs, etc.) at one or more contiguous genetic loci and then considers which haplotypes are most likely to account for the observed data. The procedure is nearly linear in the number of markers examined, and therefore more accurate and efficient.

Protein-Acrylamide Copolymer Hydrogels for Measuring Protein Concentration and Activity

UW-Madison researchers have developed a method of specifically attaching proteins to glass surfaces via copolymerization with a polyacrylamide hydrogel. They also have developed techniques for using the arrays formed using this process to detect proteins and measure their concentrations, binding affinities and kinetics.

Their method uses a surface functionalized with an acrylic acid- or acrylamide-based hydrogel. Proteins are labeled with an acrylic moiety and then attached to the functionalized surface through copolymerization with the hydrogel. The attached protein is then available for use in a variety of assays.

Fluorescence Polarization Assay to Detect Protease Cleavage

UW-Madison researchers have developed a fluorescence polarization technology to measure protease activity in real time. Fluorescent polarization can be used to estimate the size of a protein to which a fluorescent complex is attached. In the method, an uncharacterized protein is conjugated to a fluorescence tag. The fluorescent tag exhibits little fluorescence until it binds to a specific fluorescent ligand to create a highly fluorescent complex. The complex comprising the protein, fluorescent tag and fluorescent ligand is then placed in contact with a protease. When the protease cleaves the protein into two or more fragments, the fluorescence polarization of the complex decreases. The rate of change in fluorescence polarization can be measured in real time, and is equivalent to the rate of protease cleavage.

Inductive Detection for Mass Spectrometry

UW-Madison researchers have developed an enhanced electrospray ionization, time-of-flight mass spectrometer for the analysis of large or complex biomolecules. This device combines a single droplet ion source with multiple, “on-axis”, non-destructive, inductive ion detectors. The implementation of on-axis ion trajectories throughout the mass analyzer, along with multiple detectors, allows pre-acceleration ion velocities to be determined. A second set of ion detectors detects post-acceleration velocities, providing time-of-flight information while reducing ion detection losses of larger, slower-moving biomolecules. The detectors can be configured to simultaneously analyze charge states and mass-to-charge ratios, enabling the determination of absolute mass.

Input Feature and Kernel Selection Improves Speed, Accuracy and Efficiency of Support Vector Machine Classification

UW-Madison researchers have developed a selection technique that makes use of a fast Newton method to produce a reduced set of input features for linear SVM classifiers or a reduced set of kernel functions for non-linear SVM classifiers. The ability to suppress less useful data and select a reduced set of meaningful support vectors promises to increase the computational speed, accuracy and efficiency of SVM classification for data mining operations.

Droplet Ion Source for Mass Spectrometry

UW-Madison researchers have now developed a new TOF mass spectrometer that uses single charged droplets as ion sources and focuses these droplets, through use of an aerodynamic lens, onto the center axis of the mass analyzer. Discrete ion droplets are produced by ESI through the use of a novel piezoelectric dispenser (see WARF reference number P01294US). The combination of these advances promises to make mass spectrometry suitable to a wide range of previously unattainable applications, including DNA sequencing, protein identification, and quantification of relative protein expression levels.

Piezoelectric Charged Droplet Source for Mass Spectrometry

UW-Madison researchers have developed a new nanotechnology source that generates single packets of ions for mass spectrometric analysis. A sample solution held at a selected electric potential is ejected from a narrow exit aperture by constriction of a cylindrical piezoelectric element.

Unlike conventional nanoelectrospray sources that are continuous, this source dispenses discrete volumes (droplets) of electrically charged solutions as small as 10 picoliters. It also allows adjustable control of droplet exit time, ion formation time, droplet number, repetition rate and electric charge state of the droplet. When coupled to an orthogonal time-of-flight mass spectrometer, the source provides detection sensitivity in the attomole range.

New Method of Charge Reduction in Electrospray Mass Spectrometry

UW-Madison researchers have developed an improved means to reduce and control the charge states of ions generated by electrospray ionization. They previously developed a technique for reducing the charge state of ions by using a polonium alpha particle source (see WARF reference number P99352US). The technology featured here improves upon the previous one by using a corona discharge to reduce ion charge state distributions. It also allows users to produce gas-phase ions of positive or negative polarity, and which carry either a fixed charge or possess a charge state distribution that varies selectively over time.

Markerless Gene Replacement Plasmids for E. coli

UW-Madison researchers have discovered a simple and efficient gene replacement method that permits targeted introduction of markerless deletions, insertions and point mutations into the E. coli chromosome. In this method, the mutant allele is carried on a circular plasmid that integrates into the chromosome at a homologous locus, resulting in a direct duplication. Resolution of this cointegrate via intramolecular recombination is controlled by introducing a unique double-stranded break into the chromosome by the meganuclease I-SceI. The enzyme recognizes an 18-base pair sequence and generates a double-stranded break with a four-base 3` hydroxyl overhang. The method can be used in recombination-proficient E. coli and produces markerless replacements at high efficiency.

Super Bac 1 - An Inducible Vector With Shuttle Capability

UW-Madison researchers have now engineered an improved BAC vector, called Super BAC 1, for use in genomic studies. They have modified a widely used BAC to include an oriV gene from the IncP plasmid RK2. In the presence of a specific transcription factor, oriV provides arabinose-inducible replication, allowing a 50 to 150-fold amplification of BAC copy number upon addition of arabinose. As a second modification, they have added the oriT gene, also from plasmid RK2, to the BAC. This gene confers on E. coli the capacity for conjugal mating into Bacillus when the tra gene is also supplied in trans. To maintain the transferred BAC in Bacillus, the researchers have included the rep60 gene from a Bacillus plasmid. This Bacillus-specific origin of replication maintains the plasmid at five to six copies per cell in this alternate host.

Bacterial Culture Collection from an Extreme Environment in Alaska

A team of UW-Madison researchers has now created a culture collection of over 1,000 bacterial isolates from non-permafrost soil in the floodplain of the Tanana River -- an extremely cold and mineral poor environment near Fairbanks, Alaska. To obtain the largest and most diverse collection of microbes possible, the researchers employed a range of media concentrations, added soil extract to the enrichment media, and performed extended incubations at low temperatures. Preliminary screening has identified at least 5 unique isolates with good antibiotic activity. The culture collection is arrayed in 96-well culture plates with 20 percent DMSO for preservation. 

Unique, Full-Length Mouse cDNAs

A UW-Madison researcher has developed a collection of 10,000 mouse cDNA clones. Based on sequence analysis, over 90 percent of the clones are full-length.

Libraries of Bacterial Genomic DNA Isolated from Alaskan Soils

UW-Madison researchers have compiled 10 libraries of bacterial genomic DNA isolated directly from non-permafrost soils in the floodplain of the Tanana River, an extremely cold and phosphorus poor environment near Fairbanks, Alaska. The 10 libraries, including nine bacterial artificial chromosome (BAC) libraries and one fosmid library, provide more than 63,000 clones with average insert sizes ranging from 1.5 to 47 kilobases in length. The largest inserts are over 150 kilobases in length. This collection of genomic DNA complements a collection of over 1,000 bacterial cultures isolated from the same Alaskan soils.

10,000 Sequenced Mouse Genomic Clones Derived From a CpG Library

A UW-Madison researcher has developed a set of almost 10,000 sequenced mouse genomic clones derived from a library containing mouse CpG islands. Sequence analysis showed that each clone is present only one time in the set. These clones can be used in microarrays.

Bacterial Genomic Libraries from Alaskan Soils (AK 9-13 and 16)

UW-Madison researchers have compiled six libraries of bacterial genomic DNA isolated directly from non-permafrost soils in the floodplain of the Tanana River, an extremely cold and phosphorus-poor environment near Fairbanks, Alaska. The six libraries contain almost 200,000 clones with average insert sizes ranging from 5 to 30 kilobases. This collection of genomic DNA complements a collection of over 1,000 bacterial cultures and 10 additional libraries of bacterial genomic DNA isolated from the same Alaskan soils (see WARF reference numbers P03154US and P04104US).

Janthinobacterium lividium Isolates from Alaskan Soil

UW-Madison researchers have identified red- and purple-pigmented Janthinobacterium lividium isolates that have antibacterial activity against Gram-positive bacteria. These bacteria were isolated from non-permafrost soil in the floodplain of the Tanana River -- an extremely cold and mineral poor environment near Fairbanks, Alaska. The red and purple pigments, which are only produced at temperatures below 28º C, may play a role in the antibacterial activity. This collection of bacteria complements a collection of more than 1,000 bacterial cultures and 16 libraries of bacterial genomic DNA isolated from the same Alaskan soils (see links below).

Collection of Bacterial Isolates from Alaska That Do Not Grow at Temperatures at or Above 37ºC

UW-Madison researchers have developed a culture collection of bacterial isolates that do not grow at temperatures above 37ºC. These bacteria were isolated from non-permafrost soil in the floodplain of the Tanana River—an extremely cold and mineral poor environment near Fairbanks, Alaska. This collection of bacterial cultures complements a collection of more than 1,000 bacterial cultures and 16 libraries of bacterial genomic DNA isolated from the same Alaskan soils (see links below).

Collection of Genomic Fragments That Affect Quorum Sensing

This technology describes large genomic fragments cloned from Alaskan soil bacterial isolates that affect quorum sensing. A team of UW-Madison researchers collected bacterial isolates from non-permafrost soil in the floodplain of the Tanana River—an extremely cold and mineral poor environment near Fairbanks, Alaska. Large amounts of microbial DNA were isolated directly from the soil and then screened using a high throughput quorum sensing assay.

Bacterial Genomic Libraries from Alaskan Soils (AK 19, 20 and 21)

UW-Madison researchers have compiled three libraries of bacterial genomic DNA isolated directly from non-permafrost soils in the floodplain of the Tanana River, an extremely cold and phosphorus-poor environment near Fairbanks, Alaska. This collection of genomic DNA complements a collection of more than 1,000 bacterial cultures and 18 additional libraries of bacterial genomic DNA isolated from the same Alaskan soils.

Full-Length cDNA Libraries from Dioxin- and Vehicle-Treated Mice

UW-Madison researchers have isolated two full-length cDNA libraries from non-parenchymal mouse liver cells. One library was isolated from dioxin-treated mice and the other from a control group of vehicle-treated mice. These libraries could be used to determine which genes are upregulated and which are downregulated by dioxin.

Exploring Natural Products from Actinobacteria Symbionts Associated with Animals and Plants

A UW-Madison researcher has collected more than 150 unique strains of actinomycetes obtained from ants, bees, lichens, bark beetles and other macroorganisms. DNA sequencing has confirmed that these strains are novel, and thus have not been previously screened. Bioassays revealed the in vitro production of antifungal and antibacterial compounds by many of these strains.

Bacterial Genomic Libraries from Alaskan Soils (AK 22 and 23)

UW-Madison researchers have compiled two libraries of bacterial genomic DNA isolated directly from non-permafrost soils in the floodplain of the Tanana River, an extremely cold and phosphorus-poor environment near Fairbanks, Alaska. This collection of genomic DNA complements a collection of more than 1,000 bacterial cultures and 19 additional libraries of bacterial genomic DNA isolated from the same Alaskan soils.

A Saturating Population of Insertionally Mutagenized Arabidopsis thaliana Plants

UW-Madison researchers have established a large population of Arabidopsis seed lines that have been insertionally mutagenized using T-DNA and are available in very small pools. This collection provides an excellent means of obtaining knockout plants to determine the role of plant genes. The seed collection is comprised of 8000 tubes, each containing seeds from nine independently isolated plants. Each plant line has approximately 1 to 1.4 insertional mutations. The population is three- to four-fold saturated, which confers a greater than 85 percent probability of obtaining a knockout plant for an average gene size of 3-5 kb. This feature also confers a greater than 70 percent chance of getting two or more independent knockout alleles of the same gene.