Abstract: Methods and reagents are disclosed for quantitatively analyzing a set of target nucleic acid sequences. In the method a unique set of oligonucleotide probe precursors is hybridized to the target nucleic acid sequences to produce hybrids. The hybrids are processed to alter the mass of each of the oligonucleotide probe precursors in the hybrids in a target sequence-mediated reaction to produce oligonucleotide products, each of which has a unique mass that is not a result of the presence of a mass tag in the oligonucleotide product. The processing of the hybrids may involve polymerase extension or ligation. The products are analyzed by means of mass spectrometry and the results are related to the amount of the target nucleic acid sequences in the set. Kits for carrying out the above methods are also disclosed.

Abstract: The present invention provides an improved method of detecting differential expression of a gene of interest using modified nucleotides that reduce the levels of secondary structure in a nucleic acid molecule. In certain embodiments of the invention, multiple genes of interest are provided on the surface of a solid support, such as in the form of a microarray. The presence of carefully chosen unstructured nucleic acid bases (UNAs) in the samples being assayed and in the probes on the surface of the solid support provides an internal referenced measurement that is suitable for detecting the differential expression of a gene of interest in the samples. Also provided are arrays of pairs UNA probes that are capable of detecting differential expression of a particular gene of interest in two samples of nucleic acid.

Abstract: The present invention provides general affinity based methods for separating parental genetic material. Without limitation, the inventive methods may be used to separate maternal genetic material from paternal genetic material for haplotyping purposes. According to such embodiments, once the maternal genetic material has been separated from the paternal genetic material any method of SNP genotyping can be used, and the SNP genotypes will be, by definition the genetic haplotypes.

Abstract: A system for the analysis of polyionic molecules by mass spectrometry is provided. The polyionic molecule is attached to a charged tag which neutralizes some of the charge on the polyionic analyte. The formed adduct with a reduced net charge is then analyzed by mass spectrometry, and the determined molecular weight of the adduct can be used to calculate the molecular weight of the analyte. Mass spectrometric analyses of polynucleotides and proteins are particularly amenable to this method.

Abstract: Methods are disclosed for conjugating one moiety to another moiety. In the method the moieties are reacted with one another in a protic solvent. Reaction between the moieties and the protic solvent during the conjugating is negligible or reversible. A stable bond is formed between the moieties to produce a product that is not subject to &bgr;-elimination at elevated pH. Usually, one of the moieties comprises an unsaturation between two carbon atoms. One of the carbon atoms is or becomes an electrophile during the conjugating. The other of the moieties comprises a functionality reactive with the electrophile carbon atom to form a product that comprises the unsaturation. Compounds comprising both of the moieties as well as precursor molecules are also disclosed. Methods are also disclosed for determining an analyte in a sample employing compounds as described above.

Abstract: A system for the analysis of polyionic molecules by mass spectrometry is provided. The polyionic molecule is attached to a charged tag which neutralizes some of the charge on the polyionic analyte. The formed adduct with a reduced net charge is then analyzed by mass spectrometry, and the determined molecular weight of the adduct can be used to calculate the molecular weight of the analyte. Mass spectrometric analyses of polynucleotides and proteins are particularly amenable to this method.

Abstract: An adjustable nanopore is fabricated by placing the surfaces of two planar substrates in contact, wherein each substrate contains a hole having sharp corners and edges. A corner is brought into proximity with an edge to define a triangular aperture of variable area. Ionic current in a liquid solution and through the aperture is monitored as the area of the aperture is adjusted by moving one planar substrate with respect to the other along two directional axes and a rotational axis. Piezoelectric positioners can provide subnanometer repeatability in the adjustment process. The invention is useful for characterizing, cleaving, and capturing molecules, molecular complexes, and supramolecular complexes which pass through the nanopore, and provides an improvement over previous devices in which the hole size of nanopores fabricated by etching and/or redeposition is fixed after fabrication.

Abstract: A method of evaluating for the presence of a polypeptide in an analyte, using an addressable array of capture agents linked to a substrate. The analyte is exposed to the array and a set of fixed capture agents, such that the target molecules will bind to the array by means of the capture agents. After the target molecule has bound to the capture agents, it is modified using a label. The label does not interact or mark the capture agent. Kits using such arrays are further provided.

Abstract: The present invention provides a system and methods for assaying nucleic acid molecules with reduced levels of background signal and enhanced specificity and sensitivity. In particular, the present invention provides a system and methods for detecting, sorting, tracking and characterizing nucleic acid molecules using hybridization assays with reduced levels of undesirable cross hybridization and reduced levels of intramolecular secondary structure.

Abstract: A method of fabricating polynucleotide arrays includes dissolving a nucleotide monomer, oligonucleotide, or polynucleotide in a solvent containing ionic liquid and depositing the resulting solution on an array substrate. The method has particular application to fabrication of an addressable array of polynucleotides on a substrate that carries substrate bound moieties each with a hydroxyl group. The process may be repeated at specific locations on the array to elongate the polynucleotide deposited on the array.

Abstract: A method of synthesizing polynucleotides is disclosed. The method involves contacting a first nucleotide with a selected reactive group in the presence of an ionic liquid. The selected reactive group may be on a second nucleotide, a polynucleotide, or on a moiety on an insoluble substrate, for example in an oligonucleotide synthesizer.

Abstract: An adjustable nanopore is fabricated by placing the surfaces of two planar substrates in contact, wherein each substrate contains a hole having sharp corners and edges. A corner is brought into proximity with an edge to define a triangular aperture of variable area. Ionic current in a liquid solution and through the aperture is monitored as the area of the aperture is adjusted by moving one planar substrate with respect to the other along two directional axes and a rotational axis. Piezoelectric positioners can provide subnanometer repeatability in the adjustment process. The invention is useful for characterizing, cleaving, and capturing molecules, molecular complexes, and supramolecular complexes which pass through the nanopore, and provides an improvement over previous devices in which the hole size of nanopores fabricated by etching and/or redeposition is fixed after fabrication.

Abstract: A method for detecting a target moiety is disclosed. In one embodiment, a plurality of electrodes supported by a semiconductor substrate are brought into proximity with a reaction medium comprising a sample suspected of containing the target molecule. Each of the electrodes comprises at least one target probe. A plurality of cells within the semiconductor substrate are selectively addressed to apply a stimulus to each of the electrodes to activate a predetermined redox active moiety that is associated with an electrode and to detect, by means of the electrodes, corresponding responses produced as a result of the activation of the redox active moieties. The magnitude of the corresponding responses indicates the presence or absence of the target molecule in the sample.

Abstract: Methods and reagents are disclosed which provide for more sensitive, more accurate and higher through-put analyses of target nucleic acid sequences. The methods and reagents of the present invention may be generically applied to generally any target nucleic acid sequence and do not require a priori information about the presence, location or identity of mutations in the target nucleic acid sequence. The reagents of the invention are mixtures of oligonucleotide precursors having a high level of coverage and mass number complexity, and also having tags analyzable by mass spectrometry which are covalently linked to the precursors through cleavable bonds. A method is also disclosed for analyzing a target nucleic acid sequence employing the mixtures of oligonucleotide precursors having tags analyzable by mass spectrometry covalently linked to the oligonucleotide precursors through cleavable bonds, and chemical or enzymatic assays to alter the mass of the oligonucleotide precursors prior to mass spectral analysis.

Abstract: A method of evaluating for the presence of a polypeptide in an analyte, using an addressable array of capture agents linked to a substrate. The analyte is exposed to the array and a set of fixed capture agents, such that the target molecules will bind to the array by means of the capture agents. After the target molecule has bound to the capture agents, it is modified using a label. The label does not interact or mark the capture agent. Kits using such arrays are further provided.

Abstract: Methods are disclosed for conjugating one moiety to another moiety. In the method the moieties are reacted with one another in a protic solvent. Reaction between the moieties and the protic solvent during the conjugating is negligible or reversible. A stable bond is formed between the moieties to produce a product that is not subject to &bgr;-elimination at elevated pH. Usually, one of the moieties comprises an unsaturation between two carbon atoms. One of the carbon atoms is or becomes an electrophile during the conjugating. The other of the moieties comprises a functionality reactive with the electrophile carbon atom to form a product that comprises the unsaturation. Compounds comprising both of the moieties as well as precursor molecules are also disclosed. Methods are also disclosed for determining an analyte in a sample employing compounds as described above.

Abstract: Methods and reagents are disclosed which satisfy the need for more sensitive, more accurate and higher through-put analyses of target nucleic acid sequences. The methods and reagents may be generically applied to generally any target nucleic acid sequence and do not require a priori information about the presence, location or identity of mutations in the target nucleic acid sequence. The reagents of the invention are mixtures of natural and mass-modified oligonucleotide precursors having a high level of coverage and mass number complexity. A method is also disclosed for analyzing a target nucleic acid sequence employing the mixtures of natural and mass-modified oligonucleotide precursors and chemical or enzymatic assays to alter the mass of the oligonucleotide precursors prior to mass spectral analysis, generally via MALDI-TOF. The enzymatic assay may be a polymerase extension assay or a ligase assay. The kits for carrying out the methods of the invention are also disclosed.

Abstract: A sensor made of porous matrix or sol-gel glass and non-enzymatic macromolecular polymer immobilized in the sol-gel glass. The macromolecule is physically entangled or otherwise trapped, and does not leach regardless of exposure to elevated temperature and pressure. Surface effects are minimized since the there is no chemical bond between macromolecules and sol-gel glass. Indicator molecules may be attached to the macromolecular polymer either before or after the macromolecule is incorporated into the porous matrix.

Abstract: An apparatus for collecting samples from capillary electrophoresis (CE) for matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is disclosed. The apparatus has a capillary, a metallic support, a porous member, and a power supply for applying an electrical potential between the porous member and an inlet end of the capillary to drive the sample through the capillary during CE. The capillary transmits a sample from the inlet end to the exit end during capillary electrophoresis. The metallic support supports the porous membrane such that the porous membrane contacts the capillary exit end during CE. The metallic support (with the porous membrane) is suitable for placing in a mass spectrometer to act as a repeller for MALDI. The porous member has at least a portion thereof that is generally concentric with the capillary exit end and contacts the porous membrane during CE.

Abstract: A method is provided for detecting an analyte of interest in a sample. The method involves binding the analyte to the surface of a substrate through a biotin-biotin binding protein interaction, contacting the surface-bound analyte with a quantitatively detectable analyte-binding moiety that binds thereto, measuring the quantity of detectable moiety bound to the substrate surface and deriving therefrom the quantity of analyte in solution. A preferred use for the present method is in conjunction with a piezoelectric surface transverse wave device. Novel reagents useful for carrying out the inventive method are provided as well.