Nanogen, Inc.

Gamida for Life B.v.

Nanogen/becton Dickinson Partnership

Ronald George Sosnowski

Eugene Tu

Michael James Heller

Michael I Nerenberg

James P O'Connell

Carl Frederick Edman

William Frank Butler

Glen A Evans

Thomas R Jackson

David Canter

Ray R Radtkey

Ling Wang

Robert David Juncosa

Lorelei P Westin

Geoffrey C Landis

Lana L Feng

A method for electronically stabilizing hybridization of nucleic acids bound at a test site of a microelectronic device is described. First and second negatively charged nucleic acids are provided, the second nucleic acid being bound to the test site. A zwitterionic buffer having a conductance of less than 100 mS/cm is applied to the microelectronic device. A current is applied to the test site to positively bias the test site, such that the first negatively charged nucleic acid is transported to the positively biased test site having the bound the second negatively charged nucleic acid. At the test site, the first and second negatively charged nucleic acids hybridize. The zwitterionic buffer acquires a net positive charge under influence of the current, such that the positively charged zwitterionic buffer stabilizes the hybridization by reducing the repulsion between the first and second negatively charged nucleic acids.

Self-addressable self-assembling microelectronic integrated systems, component devices, mechanisms, methods, and procedures for molecular biological analysis and diagnostics

A method for analyzing nucleic acid obtained from a cell sample on a platform is described. A platform having a cell selector, a nucleic acid selector, and an array of microlocations, wherein at least one microlocation has an associated capture sequence, is provided. The cell selector is contacted with a cell sample, wherein a portion of the cells remain associated with the cell selector. At least a portion of cells associated with the cell selector are lysed to release a nucleic acid sample. The nucleic acid selector is then contacted with the nucleic acid sample, such that a portion of the nucleic acid sample remains associated with the nucleic acid selector. The associated nucleic acid sample is then released from the nucleic acid selector and then is contacted with the array of microlocations, such that at least a portion of the released nucleic acid sample hybridizes with the capture sequence.

Self-addressable self-assembling microelectronic systems and devices for molecular biological analysis and diagnostics

A system for performing molecular biological diagnosis, analysis and multistep and multiplex reactions utilizes a selfaddressable, selfassembling microelectronic system for actively carrying out controlled reactions in microscopic formats. The device includes a power supply and waveform generator adapted to supply a DC bias and superimposed AC signal to the system through an interface to the array of microlocations.

Active programmable matrix devices

This application includes methods for detecting single nucleotide polymorphisms (SNPs) in a sample using an electronically addressable microchip having a plurality of test sites. A sample nucleic acid is electronically biased, concentrated at, and immobilized to a test site on the microchip. A mixture comprising a first labeled probe and a second labeled probe is electronically hybridized to the sample nucleic acid to form first or second hybridized complexes. The first labeled probe is perfectly complementary to the first sample nucleic acid and the second labeled probe is complementary to the sample nucleic acid and contains a nucleotide that forms a mismatch with the nucleotide at the site of the polymorphism. The first or second hybridized complexes are detected by determining a signal intensity of the label of the first or second probe.

Methods for determination of single nucleic acid polymorphisms using a bioelectronic microchip

A self-addressable, self-assembling microelectronic device is designed and fabricated to actively carry out and control multi-step and multiplex molecular biological reactions in microscopic formats. These reactions include nucleic acid hybridizations, antibody/antigen reactions, diagnostics, and biopolymer synthesis. The device can be fabricated using both microlithographic and micro-machining techniques. The device can electronically control the transport and attachment of specific binding entities to specific micro-locations. The specific binding entities include molecular biological molecules such as nucleic acids and polypeptides. The device can subsequently control the transport and reaction of analytes or reactants at the addressed specific micro-locations. The device is able to concentrate analytes and reactants, remove non-specifically bound molecules, provide stringency control for DNA hybridization reactions, and improve the detection of analytes. The device can be electronically replicated.

Methods for electronically-controlled enzymatic reactions

Methods and apparatus are provided for the analysis and determination of the nature of repeat units in a genetic target. In one method of this invention, the nature of the repeat units in the genetic target is determined by the steps of providing a plurality of hybridization complex assays arrayed on a plurality of test sites, where the hybridization complex assay includes at least a nucleic acid target containing a simple repetitive DNA sequence, a capture probe having a first unique flanking sequence and n repeat units, where n&equals;0,1,2 . . . , or fractions thereof, being complementary to the target sequence, and a reporter probe having a selected sequence complementary to the same target sequence strand wherein the selected sequence of the reporter includes a second unique flanking sequence and m repeat units, where m&equals;0,1,2 . . . , or fractions thereof, but where the sum of repeat units in the capture probe plus reporter probe is greater than 0 (n&plus;m>0). Concordance and discordance among the hybridization complex assays at the test sites is determined at least in part by hybridization stability. Electronic stringency control may be utilized. Applications include paternity testing, forensic use, and disease diagnostics, such as for the identification of the existence of a clonal tumor.

Methods and apparatus for detecting variants utilizing base stacking

Methods and compositions of matter are provided for the strand displacement amplification of target nucleic acids of interest using primer pair sets that are anchored to electronically addressable capture sites on a microarray. The primer pair sets may be individually bound to the capture sites or may comprise a unique branched primer pair moiety. The anchored primers allow for the simultaneous multiplex capture, amplification and detection of a target nucleic acid derived from any sample source.

Anchored strand displacement amplification on an electronically addressable microchip

A self-addressable, self-assembling microelectronic device is designed and fabricated to actively carry out and control multi-step and multiplex molecular biological reactions in microscopic formats. These reactions include nucleic acid hybridizations, antibody/antigen reactions, diagnostics, and biopolymer synthesis. The device can be fabricated using both microlithographic and micro-machining techniques. The device can electronically control the transport and attachment of specific binding entities to specific microlocations. The specific binding entities include molecular biological molecules such as nucleic acids and polypeptides. The device can subsequently control the transport and reaction of analytes or reactants at the addressed specific microlocations. The device is able to concentrate analytes and reactants, remove non-specifically bound molecules, provide stringency control for DNA hybridization reactions, and improve the detection of analytes. The device can be electronically replicated.

Method for enhancing the hybridization efficiency of target nucleic acids using a self-addressable, self-assembling microelectronic device

Methods are provided for the analysis and determination of the nature of single nucleic acid polymorphisms (SNPs) in a genetic target. In one method of this invention, the nature of the SNPs in the genetic target is determined by the steps of providing a plurality of hybridization complexes arrayed on a plurality of test sites on an electronically bioactive microchip, where the hybridization complex includes at least a nucleic acid target containing a SNP, a stabilizer probe having a sequence complementary to the target sequence and/or reporter probe, and a reporter probe having a selected sequence complementary to either the stabilizer or the same target sequence strand wherein a selected sequence of the reporter includes either a wild type nucleotide or a nucleotide corresponding to the SNP of the target. In accordance with the invention, the stabilizer, reporter and target amplicons are hybridized using electronic assistance of the microchip system such that base-stacking energies are utilized in discerning among other identifying indicators, the presence of wild type or polymorphism sequence. Applications include disease diagnostics, such as for the identification of polymorphisms in structural genes, regulatory regions, antibiotic or chemotherapeutic resistance conferring regions, or for SNPs associated with speciation or used for determination of genetic linkage.

Methods for determination of single nucleic acid polymorphisms using bioelectronic microchip

Methods and apparatus for determination of length polymorphisms in DNA

A system for performing molecular biological diagnosis, analysis and multistep and multiplex reactions utilizes a selfaddressable, selfassembling microelectronic system for actively carrying out controlled reactions in microscopic formats. Preferably, a fluidic system flow a sample across an active area of the biochip, increasing diagnostic efficiency. Preferably, the fluidic system includes aflow cell having a window. Pulsed activation of the electrodes of the biochip are advantageously employed with the fluidic system, permitting more complete sampling of the materials within the biological sample. An improved detection system utilizes a preferably coaxially oriented excitation fiber, such as a fiber optic, disposed within a light guide, such as a liquid light guide. In this way, small geometry systems may be fluorescently imaged. A highly automated DNA diagnostic system results. Perturbation of the fluorescence signal during electronic denaturation is detailed and analyzed for analytical and diagnostic purposes. Such fluorescence perturbation information is combined with other information to provide improved analysis. DNA fingerprinting uses hybridizing DNA fragments of a given length and a capture sequence at a test site and then determining the level of reverse bias necessary to affect the hybridization, such as to de-hybridize, and determine the length of the DNA.

Method for fingerprinting utilizing an electronically addressable array

Methods for transport in molecular biological analysis and diagnostics

Methods for determining nature of repeat units in DNA

Methods and procedures for molecular biological analysis and diagnostics

Methods for electronic perturbation of fluorescence, chemilluminescence and other emissive materials provide for molecular biological analysis. In a preferred method for hybridization analysis of a sample, an electronic stringency control device is used to perform the steps of: providing the sample, a first probe with a fluorescent label and a second probe with a label under hybridization conditions on the electronic stringency control device, forming a hybridization product, subjecting the hybridization product to an electric field force, monitoring the fluorescence from the hybridization product, and analyzing the fluorescent signal. The label preferably serves as a quencher for the fluorescent label. In yet another aspect of this invention, a method for achieving electronic fluorescence perturbation on an electronic stringency control device comprising the steps of: locating a first polynucleotide and a second polynucleotide adjacent the electronic stringency control device, the first polynucleotide and second polynucleotide being complementary over at least a portion of their lengths and forming a hybridization product, the hybridization product having an associated environmental sensitive emission label, subjecting the hybridization product and label to a varying electrophoretic force, monitoring the emission from the label, and analyzing the monitored emission to determine the electronic fluorescence perturbation effect. In yet another aspect of this invention, a method is provided for electronic perturbation catalysis of substrate molecules on an electronic control device containing at least one microlocation comprising the steps of: immobilizing on the microlocation an arrangement of one or more reactive groups, exposing the reactive groups to a solution containing the substrate molecules of interest, and applying an electronic pulsing sequence which causes charge separation between the reactive groups to produce a catalytic reaction on the substrate molecules.

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Coronado, CA, United States of America

Ronald George Sosnowski