In certain embodiments, the present invention provides amplification methods in which nucleotide tag(s) and, optionally, a barcode nucleotide sequence are added to target nucleotide sequences. In other embodiments, the present invention provides a microfluidic device that includes a plurality of first input lines and a plurality of second input lines. The microfluidic device also includes a plurality of sets of first chambers and a plurality of sets of second chambers. Each set of first chambers is in fluid communication with one of the plurality of first input lines. Each set of second chambers is in fluid communication with one of the plurality of second input lines. The microfluidic device further includes a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines and a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.
Systems and associated methods and techniques for illuminating and imaging a device, such as a microfluidic or microarray device, are described herein. An optical source that illuminates the planar surface at an oblique angle can be used with optical components, such as an offset optical shaping rod and a wedge prism, used to provide uniform illumination across the planar surface and allow the illumination to appropriately reach the target illumination area despite the geometric limitations imposed by the presence and position of imaging, microfluidic control, and/or thermal cycling components.
G01N 21/00 - Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
G01N 31/22 - Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroupsApparatus specially adapted for such methods using chemical indicators
Systems and associated methods and techniques for illuminating and imaging a device, such as a microfluidic or microarray device, are described herein. An optical source that illuminates the planar surface at an oblique angle can be used with optical components, such as an offset optical shaping rod and a wedge prism, used to provide uniform illumination across the planar surface and allow the illumination to appropriately reach the target illumination area despite the geometric limitations imposed by the presence and position of imaging, microfluidic control, and/or thermal cycling components.
G01N 31/22 - Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroupsApparatus specially adapted for such methods using chemical indicators
G01N 21/00 - Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
Described herein are methods useful for incorporating one or more adaptors and/or nucleotide tag(s) and/or barcode nucleotide sequence(s) one, or typically more, target nucleotide sequences. In particular embodiments, nucleic acid fragments having adaptors, e.g., suitable for use in high-throughput DNA sequencing are generated. In other embodiments, information about a reaction mixture is encoded into a reaction product. Also described herein are methods and kits useful for amplifying one or more target nucleic acids in preparation for applications such as bidirectional nucleic acid sequencing. In particular embodiments, methods of the invention entail additionally carrying out bidirectional DNA sequencing. Also described herein are methods for encoding and detecting and/or quantifying alleles by primer extension.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12N 15/65 - Introduction of foreign genetic material using vectorsVectorsUse of hosts thereforRegulation of expression using markers
C12N 15/66 - General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligationUse of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
The present disclosure provides a “looping amplification” method to increase the specificity of nucleic acid amplification. This increased specificity facilitates multiplexing to a much higher degree than was previously possible.
C12Q 1/6853 - Nucleic acid amplification reactions using modified primers or templates
C12Q 1/6848 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
In certain embodiments, the present invention provides amplification methods in which nucleotide tag(s) and, optionally, a barcode nucleotide sequence are added to target nucleotide sequences. In other embodiments, the present invention provides a microfluidic device that includes a plurality of first input lines and a plurality of second input lines. The microfluidic device also includes a plurality of sets of first chambers and a plurality of sets of second chambers. Each set of first chambers is in fluid communication with one of the plurality of first input lines. Each set of second chambers is in fluid communication with one of the plurality of second input lines. The microfluidic device further includes a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines and a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.
Described herein are methods, kits and systems for sample enrichment, multi-step library preparation, sample normalization, detection of sample biomolecules and combinations thereof. Enrichment and multi-step library preparation is described in the context of microfluidic workflows. Sample barcoding methods and kits are described for increasing sample throughput while reducing background in negative samples. Integrated microfluidic devices comprising sample processing unit cells coupled to an array of reaction sites are provided for integrated workflows.
A microscopy apparatus comprises a microscope comprising a stage configured to hold a tissue sample, a UV laser assembly configured to emit a UV laser beam to a viewing area of the tissue sample, and an IR laser assembly configured to emit an IR laser beam to the viewing area of the tissue sample. The UV and IR laser assemblies are oriented so as to emit the respective UV and IR laser beams in a same direction.
A microscopy apparatus comprises a microscope comprising a stage configured to hold a tissue sample, a UV laser assembly configured to emit a UV laser beam to a viewing area of the tissue sample, and an IR laser assembly configured to emit an IR laser beam to the viewing area of the tissue sample. The UV and IR laser assemblies are oriented so as to emit the respective UV and IR laser beams in a same direction.
Embodiments of the present invention relate to replacement of the previous ICP-based ionisation system with a new laser ionisation system, providing improved mass spectrometer-based apparatus and methods for using them to analyse samples, in particular the use of mass spectrometry mass cytometry, imaging mass spectrometry and imaging mass cytometry, for the analysis of biological samples. Accordingly, embodiments of the present invention provide an apparatus, for example a mass cytometer, comprising: 1 ) a sampler; 2) a laser ionisation system to receive material removed from the sample by the sampler, wherein the laser ionisation system comprises an ionisation system conduit and a pulsed laser adapted to ionise sample material passing through or exiting the ionisation system conduit; and 3) a mass spectrometer to receive elemental ions from said ionisation system and to analyse said elemental ions.
G01N 27/62 - Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosolsInvestigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electric discharges, e.g. emission of cathode
G01N 21/71 - Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light thermally excited
H01J 49/04 - Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locksArrangements for external adjustment of electron- or ion-optical components
H01J 49/26 - Mass spectrometers or separator tubes
A lyophilized antibody panel is disclosed for interrogation using elemental analysis. The antibody panel includes multiple antibodies each element-tagged or element-labelled with one or more isotopes such that each different antibody is isotopically distinguishable from the other antibodies. Each element tag can include one or more unique isotopes or unique combinations of isotopes. The set of element-tagged antibodies can be lyophilized in admixture. Thus, the lyophilized element-tagged antibody panel can be easily and efficiently resuspended and mixed with a sample prior to interrogation with an elemental analyzer, such as a mass spectrometer. This lyophilized element-tagged antibody panel can provide the benefits of an element-tagged assay while also being easy to use and remaining stable for long durations.
G01N 27/62 - Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosolsInvestigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electric discharges, e.g. emission of cathode
G01N 33/50 - Chemical analysis of biological material, e.g. blood, urineTesting involving biospecific ligand binding methodsImmunological testing
As described herein, one or more parameters of a direct ionization imaging mass spectrometer (IMS) may be set to obtain a desired plasma and deliver it to a mass detector. Depending on the application, certain parameters may be predetermined (e.g., a spot size given a desired resolution) and, as described herein, other parameters can be adjusted to obtain the desired plasma properties. Also included is sample preparation suitable for direct ionization IMS and/or other imaging modalities.
C12Q 1/6872 - Methods for sequencing involving mass spectrometry
G01N 27/62 - Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosolsInvestigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electric discharges, e.g. emission of cathode
G01N 33/48 - Biological material, e.g. blood, urineHaemocytometers
H01J 49/00 - Particle spectrometers or separator tubes
The invention relates to systems and methods for autofocusing in imaging mass spectrometry. The present invention provides improvements over current IMS and IMG apparatus and methods through an autofocus component including plurality of apertures in the autofocus system, such as a plurality of apertures arranged in 2 dimensions. As a plurality of apertures is used the autofocus system provides redundancy in the event that measurement of focus on the sample from the illuminating radiation passed through one or more of the apertures fails so reducing the number of unsuccessful autofocus attempts.
H01J 49/04 - Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locksArrangements for external adjustment of electron- or ion-optical components
This disclosure relates to systems and methods for high speed modulation sample imaging. Disclosed herein are systems and methods for performing imaging mass cytometry, including analysis of labelling atoms by elemental (e.g., atomic) mass spectrometry. Aspects include a sampling system having, and method of using, a femtosecond (fs) laser and/or laser scanning. Alternatively or in addition, aspects include systems and methods for co-registering other imaging modalities with imaging mass cytometry.
H01J 49/04 - Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locksArrangements for external adjustment of electron- or ion-optical components
The present invention relates to the high resolution imaging of samples using imaging mass spectrometry (IMS) and to the imaging of biological samples by imaging mass cytometry (IMCTM) in which labelling atoms are detected by IMS. LA-ICP-MS (a form of IMS in which the sample is ablated by a laser, the ablated material is then ionised in an inductively coupled plasma before the ions are detected by mass spectrometry) has been used for analysis of various substances, such as mineral analysis of geological samples, analysis of archaeological samples, and imaging of biological substances. However, traditional LA-ICP-MS systems and methods may not provide high resolution. Described herein are methods and systems for high resolution IMS and IMC.
Embodiments of the present invention relate to reagents and their use for elemental imaging mass spectrometry of biological samples. The embodiments comprising methods for quantifying one or more analytes within a sample, comprising the steps of: (a) providing the sample, wherein the one or more analytes are immobilized to a sample carrier, wherein the sample has been labelled with one or more mass tags comprising one or more labelling atoms, (b) performing mass cytometry on the sample to determine the level of the one or more labelling atoms, wherein the level of the one or more labelling atoms corresponds to the copy number of the one or more analytes.
Analyzing samples injected into an inductively coupled plasma source can be improved by one or more of a stabilizing solution mixable with a sample prior to injection and a heated injector. The stabilizing solution can minimize the difference in osmotic pressure between the solution and the cells with a relatively low amount of dissolved solids (e.g., at or below about 0.2%). The stabilizing solution can contain a salt (e.g., ammonium nitrate) present in concentrations of at least 5 mM. The injector can be heated before and/or during injection. In some cases, heat from adjacent parts can be channeled into the injector to improve heating of the injector. An injector heated to sufficient temperatures can minimize solute buildup and can extend the usable time between cleanings. These improvements can be especially useful in elemental analysis, such as inductively coupled plasma mass spectrometry or inductively coupled plasma optical emission spectrometry.
G01N 33/50 - Chemical analysis of biological material, e.g. blood, urineTesting involving biospecific ligand binding methodsImmunological testing
G01N 33/532 - Production of labelled immunochemicals
G01N 33/543 - ImmunoassayBiospecific binding assayMaterials therefor with an insoluble carrier for immobilising immunochemicals
G01N 33/544 - ImmunoassayBiospecific binding assayMaterials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
G01N 33/566 - ImmunoassayBiospecific binding assayMaterials therefor using specific carrier or receptor proteins as ligand binding reagent
G01N 33/58 - Chemical analysis of biological material, e.g. blood, urineTesting involving biospecific ligand binding methodsImmunological testing involving labelled substances
Described herein are cell-based analytic methods, including a method of incorporating nucleic acid sequences into reaction products from a cell population, wherein the nucleic acid sequences are incorporated into the reaction products of each cell individually or in small groups of cells individually. Also described herein is a matrix-type microfluidic device that permits at least two reagents to be delivered separately to each cell or group of cells, as well as primer combinations useful in the method and device.
Disclosed herein is a method and kit thereof for isolating nucleic acids from wax-embedded samples using hexadecane as the solvent to dissolve the sample, precipitating and washing the extracted nucleic acids using ethanol, and dissolving the nucleic acids in a lysis buffer that includes NP40 and SDS. By implementing the reagents and techniques described in this disclosure, the user can obtain a product that has better yield, less degradation, and contains more unique mRNA transcripts for subsequent sequencing and analysis.
Disclosed herein is a method and kit thereof for isolating nucleic acids from wax-embedded samples using hexadecane as the solvent to dissolve the sample, precipitating and washing the extracted nucleic acids using ethanol, and dissolving the nucleic acids in a lysis buffer that includes NP40 and SDS. By implementing the reagents and techniques described in this disclosure, the user can obtain a product that has better yield, less degradation, and contains more unique mRNA transcripts for subsequent sequencing and analysis.
Described herein are methods for preparing DNA templates for single-cell transcript sequencing of RNA from a population of cells. The methods entail distributing cells from the population into separate reaction volumes so that a plurality of separate reaction volumes each contain a single, isolated cell, wherein the cells have been treated with a fixative prior to distribution. The isolated cells are then permeabilized or disrupted, and cDNA is prepared by reverse transcript, followed by amplification. Also provided is a novel chemistry for efficient production of DNA templates from T-cell receptors or immunoglobulins in single cells.
Described herein are methods for preparing DNA templates for single-cell transcript sequencing of RNA from a population of cells. The methods entail distributing cells from the population into separate reaction volumes so that a plurality of separate reaction volumes each contain a single, isolated cell, wherein the cells have been treated with a fixative prior to distribution. The isolated cells are then permeabilized or disrupted, and cDNA is prepared by reverse transcript, followed by amplification. Also provided is a novel chemistry for efficient production of DNA templates from T-cell receptors or immunoglobulins in single cells.
In certain embodiments, the present invention provides amplification methods in which nucleotide tag(s) and, optionally, a barcode nucleotide sequence are added to target nucleotide sequences. In other embodiments, the present invention provides a microfluidic device that includes a plurality of first input lines and a plurality of second input lines. The microfluidic device also includes a plurality of sets of first chambers and a plurality of sets of second chambers. Each set of first chambers is in fluid communication with one of the plurality of first input lines. Each set of second chambers is in fluid communication with one of the plurality of second input lines. The microfluidic device further includes a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines and a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.
The present disclosure provides a "looping amplification" method to increase the specificity of nucleic acid amplification. This increased specificity facilitates multiplexing to a much higher degree than was previously possible.
The present disclosure provides a “looping amplification” method to increase the specificity of nucleic acid amplification. This increased specificity facilitates multiplexing to a much higher degree than was previously possible.
C12Q 1/6853 - Nucleic acid amplification reactions using modified primers or templates
C12Q 1/6848 - Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
The present disclosure provides a "looping amplification" method to increase the specificity of nucleic acid amplification. This increased specificity facilitates multiplexing to a much higher degree than was previously possible.
The present invention provides methods for analysis of genomic DNA and/or RNA from small samples or even single cells. Methods for analyzing genomic DNA can entail whole genome amplification (WGA), followed by preamplification and amplification of selected target nucleic acids. Methods for analyzing RNA can entail reverse transcription of the desired RNA, followed by preamplification and amplification of selected target nucleic acids.
Described herein are cell-based analytic methods, including a method of incorporating nucleic acid sequences into reaction products from a cell population, wherein the nucleic acid sequences are incorporated into the reaction products of each cell individually or in small groups of cells individually. Also described herein is a matrix-type microfluidic device that permits at least two reagents to be delivered separately to each cell or group of cells, as well as primer combinations useful in the method and device.
Described herein are cell-based analytic methods, including a method of incorporating nucleic acid sequences into reaction products from a cell population, wherein the nucleic acid sequences are incorporated into the reaction products of each cell individually or in small groups of cells individually. Also described herein is a matrix-type microfluidic device that permits at least two reagents to be delivered separately to each cell or group of cells, as well as primer combinations useful in the method and device.
C40B 20/00 - Methods specially adapted for identifying library members
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
C40B 40/06 - Libraries containing nucleotides or polynucleotides, or derivatives thereof
C40B 50/06 - Biochemical methods, e.g. using enzymes or whole viable microorganisms
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
30.
SINGLE-CELL NUCLEIC ACIDS FOR HIGH-THROUGHPUT STUDIES
Described herein are cell-based analytic methods, including a method of incorporating nucleic acid sequences into reaction products from a cell population, wherein the nucleic acid sequences are incorporated into the reaction products of each cell individually or in small groups of cells individually. Also described herein is a matrix-type microfluidic device that permits at least two reagents to be delivered separately to each cell or group of cells, as well as primer combinations useful in the method and device.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
C12Q 1/00 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C12Q 1/6876 - Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
C40B 20/00 - Methods specially adapted for identifying library members
C40B 20/04 - Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
C40B 30/06 - Methods of screening libraries by measuring effects on living organisms, tissues or cells
C40B 40/06 - Libraries containing nucleotides or polynucleotides, or derivatives thereof
C40B 50/06 - Biochemical methods, e.g. using enzymes or whole viable microorganisms
C40B 70/00 - Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or barcodes
Described herein are methods useful for incorporating one or more adaptors and/or nucleotide tag(s) and/or barcode nucleotide sequence(s) one, or typically more, target nucleotide sequences. In particular embodiments, nucleic acid fragments having adaptors, e.g., suitable for use in high-throughput DNA sequencing are generated. In other embodiments, information about a reaction mixture is encoded into a reaction product. Also described herein are methods and kits useful for amplifying one or more target nucleic acids in preparation for applications such as bidirectional nucleic acid sequencing. In particular embodiments, methods of the invention entail additionally carrying out bidirectional DNA sequencing. Also described herein are methods for encoding and detecting and/or quantifying alleles by primer extension.
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12N 15/65 - Introduction of foreign genetic material using vectorsVectorsUse of hosts thereforRegulation of expression using markers
C12N 15/66 - General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligationUse of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
In certain embodiments, the present invention provides a way of "digitally" marking different the alleles of different chromosomes by using a transposase to insert differently barcoded transposons into genomic DNA before further analysis. According to this method, each allele becomes marked with a unique pattern of transposon barcodes. Because each unique pattern of transposon barcodes identifies a particular allele, the method facilitates determinations of ploidy and copy number variation, improves the ability to discriminate among homozygotes, heterozygotes, and patterns arising from sequencing errors, and allows loci separated by uninformative stretches of DNA to be identified as linked loci, thereby facilitating haplotype determinations. Also provided is a novel artificial transposon end that includes a barcode sequence in two or more positions that are not essential for transposition.
This disclosure provides a method of forming tagged nucleic acid sequences. A target polynucleotide is immobilized on a solid support; a recognition-oligonucleotide is hybridized thereto; the recognition-oligonucleotide-target polynucleotide hybrid is cleaved; and an adapter nucleic acid is ligated to the cleaved target polynucleotide, thereby forming a tagged nucleic acid sequence. Also provided is a method of forming a tagged single stranded cDNA; a method of forming a plurality of tagged heterogeneous nucleic acid sequences; a library of recognition-oligonucleotides; and methods for amplifying a cDNA sequence immobilized on a solid support. These methods and products can be used alone or in combination for integrated single cell sequencing, and can be adapted for use in a microfluidic apparatus or device.
Methods and reagents for detection and analysis of nucleic acids are provided. The methods employ proximity extension assays for detection of a target nucleic acids of interest, e.g., a target RNA. The method can additionally be used in multiplex assays with a protein proximity extension assay to detect protein.
Methods for cell analysis are provided, comprising cell capturing, characterization, transport, and culture. In an exemplary method individual cells (and/or cellular units) are flowed into a microfluidic channel, the channel is partitioned into a plurality of contiguous segments, capturing at least one cell in at least one segment, A characteristic of one or more captured cells is determined and the cell(s) and combinations of cells are transported to specified cell holding chamber(s) based on the determined characteristic(s). Also provided are devices and systems for cell analysis.
In certain embodiments, the present invention provides amplification methods in which nucleotide tag(s) and, optionally, a barcode nucleotide sequence are added to target nucleotide sequences. In other embodiments, the present invention provides a microfluidic device that includes a plurality of first input lines and a plurality of second input lines. The microfluidic device also includes a plurality of sets of first chambers and a plurality of sets of second chambers. Each set of first chambers is in fluid communication with one of the plurality of first input lines. Each set of second chambers is in fluid communication with one of the plurality of second input lines. The microfluidic device further includes a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines and a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C07H 21/02 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
B01L 3/00 - Containers or dishes for laboratory use, e.g. laboratory glasswareDroppers
B01L 7/00 - Heating or cooling apparatusHeat insulating devices
The present invention provides methods for analysis of genomic DNA and/or RNA from small samples or even single cells. Methods for analyzing genomic DNA can entail whole genome amplification (WGA), followed by preamplification and amplification of selected target nucleic acids. Methods for analyzing RNA can entail reverse transcription of the desired RNA, followed by preamplification and amplification of selected target nucleic acids.
The invention provides a method for detecting a target nucleotide sequence by tagging the nucleotide sequence with a nucleotide tag, providing a probe oligonucleotide with a melting temperature Tm1, comprising a regulatory sequence and a nucleotide tag recognition sequence; incorporating the probe oligonucleotide into the tagged polynucleotide in a polynucleotide amplification reaction, providing a regulatory oligonucleotide with a melting temperature Tm2, comprising a sequence segment that complementary to the regulatory sequence and a tail segment that does not hybridize to the probe nucleotide when the sequence segment and the regulatory sequence are annealed, amplifying the tagged target nucleic acid sequence in a PCR amplification reaction using the probe oligonucleotide as a primer, and using a DNA polymerase with high strand displacement activity and low 5-nuclease activity, and detecting the amplification product; wherein Tm1 and Tm2 are higher than the annealing temperature associated with the polynucleotide amplification reaction.
This invention provides technology for transdifferentiating cells from one cell type to another. The cells are cultured with one or more vector-free gene regulator oligonucleotides concurrently or in succession, and then harvested when cell markers or the morphology of the culture shows that transdifferentiation is complete. Suitable gene regular oligonucleotides include microRNAs and messenger RNAs that encode a differentiation factor. Conditions for transdifferentiation can be optimized by dividing cells into different culture chambers of a microfluidic device. Cells are cultured with different additives in each chamber, and then compared. Transdifferentiated cells produced according to this invention can provide a consistent source of tissue for use in regenerative medicine.
In certain embodiments, the invention provides methods and devices for assaying single particles in a population of particles, wherein at least two parameters are measured for each particle. One or more parameters can be measured while the particles are in the separate reaction volumes. Alternatively or in addition, one or more parameters can be measured in a later analytic step, e.g., where reactions are carried out in the separate reaction volumes and the reaction products are recovered and analyzed. In particular embodiments, one or more parameter measurements are carried out “in parallel,” i.e., essentially simultaneously in the separate reaction volumes.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
C12M 1/34 - Measuring or testing with condition measuring or sensing means, e.g. colony counters
C12M 3/00 - Tissue, human, animal or plant cell, or virus culture apparatus
C07H 21/02 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
C07H 21/00 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
B01D 15/08 - Selective adsorption, e.g. chromatography
In certain embodiments, the invention provides methods and devices for assaying single particles in a population of particles, wherein at least two parameters are measured for each particle. One or more parameters can be measured while the particles are in the separate reaction volumes. Alternatively or in addition, one or more parameters can be measured in a later analytic step, e.g., where reactions are carried out in the separate reaction volumes and the reaction products are recovered and analyzed. In particular embodiments, one or more parameter measurements are carried out "in parallel," i.e., essentially simultaneously in the separate reaction volumes.
In certain embodiments, the invention provides methods and devices for assaying single particles in a population of particles, wherein at least two parameters are measured for each particle. One or more parameters can be measured while the particles are in the separate reaction volumes. Alternatively or in addition, one or more parameters can be measured in a later analytic step, e.g., where reactions are carried out in the separate reaction volumes and the reaction products are recovered and analyzed. In particular embodiments, one or more parameter measurements are carried out "in parallel," i.e., essentially simultaneously in the separate reaction volumes.
Methods, systems, and devices are described for multiple single-cell capturing and processing utilizing microfluidics. Tools and techniques are provided for capturing, partitioning, and/or manipulating individual cells from a larger population of cells along with generating genetic information and/or reactions related to each individual cell. Different capture configurations may be utilized to capture individual cells and then processing each individual cell in a multi-chamber reaction configuration. Some embodiments may provide for specific target amplification, whole genome amplification, whole transcriptome amplification, real-time PCR preparation, copy number variation, preamplification, mRNA sequencing, and/or haplotyping of the multiple individual cells that have been partitioned from the larger population of cells. Some embodiments may provide for other applications. Some embodiments may be configured for imaging the individual cells or associated reaction products as part of the processing. Reaction products may be harvested and/or further analyzed in some cases.
A microfluidic device includes an input source characterized by a source pressure and an input channel in fluid communication with the input source. The microfluidic device also includes an output channel and a valve having an open state and a closed state. The valve is disposed between the input channel and the output channel and is characterized by a static pressure. The microfluidic device further includes a control channel coupled to the valve and characterized by a control pressure. In the closed state, the control pressure is greater than atmospheric pressure.
Described herein are methods useful for incorporating one or more adaptors and/or nucleotide tag(s) and/or barcode nucleotide sequence(s) one, or typically more, target nucleotide sequences. In particular embodiments, nucleic acid fragments having adaptors, e.g., suitable for use in high-throughput DNA sequencing are generated. In other embodiments, information about a reaction mixture is encoded into a reaction product. Also described herein are methods and kits useful for amplifying one or more target nucleic acids in preparation for applications such as bidirectional nucleic acid sequencing. In particular embodiments, methods of the invention entail additionally carrying out bidirectional DNA sequencing. Also described herein are methods for encoding and detecting and/or quantifying alleles by primer extension.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C12N 15/10 - Processes for the isolation, preparation or purification of DNA or RNA
C12N 15/65 - Introduction of foreign genetic material using vectorsVectorsUse of hosts thereforRegulation of expression using markers
C12N 15/66 - General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligationUse of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
Described herein are methods useful for incorporating one or more adaptors and/or nucleotide tag(s) and/or barcode nucleotide sequence(s) one, or typically more, target nucleotide sequences. In particular embodiments, nucleic acid fragments having adaptors, e.g., suitable for use in high-throughput DNA sequencing are generated. In other embodiments, information about a reaction mixture is encoded into a reaction product. Also described herein are methods and kits useful for amplifying one or more target nucleic acids in preparation for applications such as bidirectional nucleic acid sequencing. In particular embodiments, methods of the invention entail additionally carrying out bidirectional DNA sequencing. Also described herein are methods for encoding and detecting and/or quantifying alleles by primer extension.
The invention provides a method for detecting a target nucleotide sequence by tagging the nucleotide sequence with a nucleotide tag, providing a probe oligonucleotide with a melting temperature Tm1, comprising a regulatory sequence and a nucleotide tag recognition sequence; incorporating the probe oligonucleotide into the tagged polynucleotide in a polynucleotide amplification reaction, providing a regulatory oligonucleotide with a melting temperature Tm2, comprising a sequence segment that is at least partially complementary to the regulatory sequence, amplifying the tagged target nucleic acid sequence in a PCR amplification reaction using the probe oligonucleotide as a primer, and detecting the amplification product; wherein Tm1 and Tm2 are higher than the annealing temperature associated with the polynucleotide amplification reaction.
Methods and reagents for detection and analysis of nucleic acids are provided. Certain methods involves an encoding amplification in which a target sequence is associated with probe-binding sequences and optionally with indexing sequences, (2) an optional distribution step in which the product of the encoding amplification is split into multiple aliquots, and (3) a decoding and detection step in which the presence, absence, quantity, or relative amount of the target sequence in the aliquots is determined. The detection step makes use of a multifunctional "self-digesting" molecular probe comprising a primer polynucleotide and a probe oligonucleotide, linked in a 5'-5' orientation.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C07H 21/00 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
Reagents and methods are provided for detecting the presence of a target polynucleotide in a sample are disclosed. In one aspect, a method for producing a labeled amplification product by amplifying a target nucleic acid sequence to produce an amplification product comprising the target sequence, a first probe-binding sequence 5' to the target sequence, and a second probe-binding sequence 3' to the target sequence, thereby producing an amplification product; and hybridizing a first detection probe to the amplification product, said first detection probe comprising a first segment that hybridizes to the first probe-binding sequence and a second segment that hybridizes to the second probe-binding sequence, thereby producing a labeled amplification product is disclosed.
The present invention provides amplification-based methods for detection of genotype, mutations, and/or aneuploidy. These methods have broad applicability, but are particularly well-suited to detecting and quantifying target nucleic acids in free fetal DNA present in a maternal bodily fluid sample.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
G01N 33/52 - Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper
G06F 19/18 - for functional genomics or proteomics, e.g. genotype-phenotype associations, linkage disequilibrium, population genetics, binding site identification, mutagenesis, genotyping or genome annotation, protein-protein interactions or protein-nucleic acid interactions
The present invention provides methods for selectively enriching a biological sample for short nucleic acids, such as fetal DNA in a maternal sample or apoptic DNA in a biological sample from a cancer patient and for subsequently analyzing the short nucleic acids for genotype, mutation, and/or aneuploidy.
Provided are cartridges and systems for effecting automated extraction, isolation, and purification of cellular components - such as nucleic acids - from a cellular sample in assay-ready form. Also provided are related methods of effecting such sample processing.
Kits, primers, and methods are provided herein for detecting relative target source to reference source ratios in a biological sample, by distributing the biological sample into discrete subsamples, wherein the biological sample includes, a plurality of target molecules on a target source; and a plurality of reference molecules on a reference source; providing target primers directed to one or more of the plurality of target molecules and reference primers directed to one or more of the plurality of reference molecules; performing digital amplification with the target primers and the reference primers; and detecting the presence or absence of amplified target products with target probes and detecting the presence or absence of amplified reference products with reference probes, wherein the ratio of amplified target products to amplified reference products is indicative of a relative amount of target source to reference source in a biological sample.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
G01N 33/50 - Chemical analysis of biological material, e.g. blood, urineTesting involving biospecific ligand binding methodsImmunological testing
A method for rendering a microfluidic device suitable for reuse for nucleic acid analysis is provided. The method may include flowing a nucleic acid inactivating solution into a microfluidic channel of the device by pumping; and then flowing a wash solution into the channel by pumping, thereby displacing the nucleic acid inactivating solution from the channel, whereby any residual nucleic acid from a prior use of the device is inactivated.
The invention relates to methods, reagents and devices for detection and characterization of nucleic acids, cells, and other biological samples. Assay method are provided in which a sample is partitioned into sub-samples, and analysis of the contents of the sub-samples carried out. The invention also provides microfluidic devices for conducting the assay. The invention also provides an analysis method using a universal primers and probes for amplification and detection.
Methods for detecting chromosomal aneuploidy of a specified chromosome or chromosome region are provided. Also provided are methods for genetic analysis of heterogeneously sized chromosomal DNA fragments. The methods are useful for non-invasive prenatal diagnosis and other genetic analyses.
The present invention includes microfluidic systems having a microfabricated cavity that may be covered with a removable cover, where the removable cover allows at least part of the opening of the microfabricated cavity to be exposed or directly accessed by an operator. The microfluidic systems comprise chambers, flow and control channels formed in elastomeric layers that may comprise PDMS. The removable cover comprises a thermoplastic base film bonded to an elastomer layer by an adhesive layer. When the removable cover is peeled off, the chamber is at least partially open to allow sample extraction from the chamber. The chamber may have macromolecular crystals formed inside or resulting contents from a PCR reaction. The invention also includes a method for making vias in elastomeric layers by using the removable cover. The invention further includes methods and devices for peeling the peelable cover or a removable component such as Integrated Heater Spreader.
G01N 35/08 - Automatic analysis not limited to methods or materials provided for in any single one of groups Handling materials therefor using a stream of discrete samples flowing along a tube system, e.g. flow injection analysis
G01N 33/48 - Biological material, e.g. blood, urineHaemocytometers
61.
Multi-primer amplification method for barcoding of target nucleic acids
In certain embodiments, the invention provides amplification methods in which nucleotide tag(s) and a barcode nucleotide sequence are added to target nucleotide sequences. In other embodiments, the present invention provides a microfluidic device that includes a plurality of first input lines and a plurality of second input lines. The microfluidic device also includes a plurality of sets of first chambers and a plurality of sets of second chambers. Each set of first chambers is in fluid communication with one of the plurality of first input lines. Each set of second chambers is in fluid communication with one of the plurality of second input lines. The microfluidic device further includes a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines and a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
C07H 21/02 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
62.
MULTI-PRIMER AMPLIFICATION METHOD FOR BARCODING OF TARGET NUCLEIC ACIDS
In certain embodiments, the present invention provides amplification methods in which nucleotide tag(s) and, optionally, a barcode nucleotide sequence are added to target nucleotide sequences. In other embodiments, the present invention provides a microfluidic device that includes a plurality of first input lines and a plurality of second input lines. The microfluidic device also includes a plurality of sets of first chambers and a plurality of sets of second chambers. Each set of first chambers is in fluid communication with one of the plurality of first input lines. Each set of second chambers is in fluid communication with one of the plurality of second input lines. The microfluidic device further includes a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines and a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.
C40B 20/04 - Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
C40B 70/00 - Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or barcodes
G01N 33/50 - Chemical analysis of biological material, e.g. blood, urineTesting involving biospecific ligand binding methodsImmunological testing
63.
MULTI-PRIMER AMPLIFICATION METHOD FOR BARCODING OF TARGET NUCLEIC ACIDS
In certain embodiments, the present invention provides amplification methods in which nucleotide tag(s) and, optionally, a barcode nucleotide sequence are added to target nucleotide sequences. In other embodiments, the present invention provides a microfluidic device that includes a plurality of first input lines and a plurality of second input lines. The microfluidic device also includes a plurality of sets of first chambers and a plurality of sets of second chambers. Each set of first chambers is in fluid communication with one of the plurality of first input lines. Each set of second chambers is in fluid communication with one of the plurality of second input lines. The microfluidic device further includes a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines and a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.
Methods are provided for selective tagging of short nucleic acids comprising a short target nucleotide sequence over longer nucleic acids comprising the same target nucleotide sequence. The methods can involve performing one or two cycles of amplification of a sample comprising long nucleic acids and short nucleic acids, each comprising the same target nucleotide sequence with at least two target-specific primers or primer pairs under suitable annealing conditions, wherein the primer pairs comprise: an inner primer or primer pair that can amplify the target nucleotide sequence on long and short nucleic acids (wherein each inner primer comprises a 5' nucleotide tag; and an outer primer or primer pair that amplifies the target nucleotide sequence on long nucleic acids, but not on short nucleic acids); whereby the amplification after a second cycle produces at least one tagged target nucleotide sequence that comprises two nucleotide tags, one from each inner primer, with the target nucleotide sequence located between the nucleotide tags.
In certain embodiments, the present invention provides amplification methods in which nucleotide tag(s) and, optionally, a barcode nucleotide sequence are added to target nucleotide sequences. In other embodiments, the present invention provides a microfluidic device that includes a plurality of first input lines and a plurality of second input lines. The microfluidic device also includes a plurality of sets of first chambers and a plurality of sets of second chambers. Each set of first chambers is in fluid communication with one of the plurality of first input lines. Each set of second chambers is in fluid communication with one of the plurality of second input lines. The microfluidic device further includes a plurality of first pump elements in fluid communication with a first portion of the plurality of second input lines and a plurality of second pump elements in fluid communication with a second portion of the plurality of second input lines.
The present invention provides for determining relative copy number difference for one or more target nucleic acid sequences between a test sample and a reference sample or reference value derived therefrom. The methods facilitate the detection of copy number differences less than 1.5-fold.
The present invention provides methods for analysis of genomic DNA and/or RNA from small samples or even single cells. Methods for analyzing genomic DNA can entail whole genome amplification (WGA), followed by preamplification and amplification of selected target nucleic acids. Methods for analyzing RNA can entail reverse transcription of the desired RNA, followed by preamplification and amplification of selected target nucleic acids.
A microfluidic device includes a pressure source and a control line in fluid communication with the pressure source. The microfluidic device also includes a plurality of valves operated via the control line and an independent valve positioned adjacent the control line and between the pressure source and the plurality of valves.
The present invention provides assay methods that increase the number of samples and/or target nucleic acids that can be analyzed in a single assay. In certain embodiments, an assay method entails separately subjecting S samples to an encoding reaction that produces a set of T tagged target nucleotide sequences, each tagged target nucleotide sequence including a sample-specific nucleotide tag and a target nucleotide sequence. In some embodiments, an assay method entails separately subjecting S samples to an encoding reaction that produces a set of T tagged target nucleotide sequences, each tagged target nucleotide sequence including a first nucleotide tag linked to a target nucleotide sequence, which is linked to a second nucleotide tag. In either case, the tagged target nucleotide sequences from the S samples can be mixed to form an assay mixture and subsequently assayed.
Microfluidic devices are described that include a rigid base layer, and an elastomeric layer on the base layer. The elastomeric layer may include at least part of a fluid channel for transporting a liquid reagent, and a vent channel that accepts gas diffusing through the elastomeric layer from the flow channel and vents it out of the elastomeric layer. The devices may also include a mixing chamber fluidly connected to the fluid channel, and a control channel overlapping with a deflectable membrane that defines a portion of the flow channel, where the control channel may be operable to change a rate at which the liquid reagent flows through the fluid channel. The devices may further include a rigid plastic layer on the elastomeric layer.
An integrated fluidic chip includes a substrate defined by a lateral surface area greater than 28 square inches. The integrated fluidic chip also includes a first elastomeric layer having a mold surface and a top surface. The mold surface of the first elastomeric layer is joined to a portion of the substrate. The first elastomeric layer includes a plurality of first channels extending normally from the substrate to a first dimension inside the first elastomeric layer. The integrated fluidic chip further includes a second elastomeric layer having a mold surface and a top surface. The mold surface of the second elastomeric layer is joined to at least a portion of the top surface of the first elastomeric layer.
Multilevel microfluidic devices include a control line that can simultaneously actuate valves for both sample and reagent lines. Microfluidic devices are configured to contain a first reagent in a first chamber and a second reagent in a second chamber, where either or both of the first and second reagents are contained at a desired or selected pressure. Operation of a microfluidic device includes transmitting second reagent from the second chamber to the first chamber, for mixing or contact with the first reagent. Microfluidic device features such as channels, valves, chambers, can be at least partially contained, embedded, or formed by or within one or more layers or levels of an elastomeric block.
A carrier for holding a microfluidic device includes a substrate with a plurality of wells, each well defining a volume of between 0.1 µl and 100 µl; a plurality of channels within the substrate wherein each well is in fluid communication with at least one of the plurality of channels; and a receiving portion for receiving a microfluidic device and placing the microfluidic device in fluid communication with the plurality of wells. The carrier has a polymeric composition and/or an array of structural features that enhance its performance and compatibility with existing instrumentation.
High throughput methods are used that combine the features of using a matrix-type microfluidic device, labeled nucleic acid probes, and homogenous assays to detect and/or quantify nucleic acid analytes. The high throughput methods are capable of detecting nucleic acid analyes with high PCR and probe specificity, producing a low fluorescence background and therefore, a high signal to noise ratio. Additionally, the high throughput methods are capable of detecting low copy number nucleic acid analyte per cell.
C07H 21/02 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
C07H 19/04 - Heterocyclic radicals containing only nitrogen as ring hetero atom
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C12P 19/34 - Polynucleotides, e.g. nucleic acids, oligoribonucleotides
77.
COPY NUMBER VARIATION DETERMINATION, METHODS AND SYSTEMS
The present invention methods and systems for determining copy number variation of a target polynucleotide in a genome of a subject including amplification based techniques. Methods can include pre-amplification of the sample followed by distribution of sample and a plurality of reaction volumes, quantitative detection of a target polynucleotide and a reference polynucleotide, and analysis so as to determine the relative copy number of the target polynucleotide sequence in the genome of the subject.
C07H 21/00 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
The disclosure provides nucleotide analogs and methods of their use. Analogs of the invention comprise a reporter molecule (label) attached via the N4, N6, O4, or O6 position of the nitrogenous base portion of the analog. In a preferred embodiment, nucleotide analogs of the invention comprise a label attached to the nitrogenous base portion of the analog via a cleavable linker at the N4, O4, N6 or O6 position.
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
C12Q 1/68 - Measuring or testing processes involving enzymes, nucleic acids or microorganismsCompositions thereforProcesses of preparing such compositions involving nucleic acids
C07H 19/04 - Heterocyclic radicals containing only nitrogen as ring hetero atom
80.
METHOD AND SYSTEM FOR CRYSTALLIZATION AND X-RAY DIFFRACTION SCREENING
An integrated fluidic circuit includes a substrate layer and a first structure coupled to the substrate layer and including a plurality of channels. The first structure is configured to provide for flow of one or more materials through the plurality of channels. The integrated fluidic circuit also includes a second structure coupled to the substrate layer. The second structure includes a plurality of control channels configured to receive an actuation pressure. The integrated fluidic circuit is characterized by a thickness of less than 1.5 mm.
The invention provides a family of tethered nucleotide analogs useful in sequencing nucleic acids containing a homopolymer region comprising, for example, two or more base repeats, and to sequencing methods using such tethered nucleotide analogs.
C07H 21/04 - Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
Disclosed are methods for performing aptamer preselection based on unique geometry and the content of stems or loops of the aptamer, which methods are capable of providing suitable binders and also permit selection of aptamers performed essentially entirely on a chip or other device. Also disclosed are kits for aptamer selection.
C40B 20/00 - Methods specially adapted for identifying library members
C40B 30/04 - Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
C40B 20/08 - Direct analysis of the library members per se by physical methods, e.g. spectroscopy
C12N 15/115 - Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith
C40B 40/08 - Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
New high density microfluidic devices and methods provide precise metering of fluid volumes and efficient mixing of the metered volumes. A first solution is introduced into a segment of a flow channel in fluidic communication with a reaction chamber. A second solution is flowed through the segment so that the first solution is displaced into the reaction chamber, and a volume of the second solution enters the chamber. The chamber can then be isolated and reactions within the chamber can be initiated and/or detected. High throughput methods of genetic analysis can be carried out with greater accuracy than previously available.
A method of adjusting amplification curves in a PCR experiment includes receiving a plurality of amplification curves for a sample and computing a first parameter for each of the plurality of amplification curves. The method also includes computing a second parameter for each of the plurality of amplification curves and computing a third parameter using at least a portion of the first or second parameters. The method further includes computing an offset for each of the plurality of amplification curves. The offset is a function of the first parameter and the third parameter. Moreover, the method includes adjusting at least one of the plurality of amplification curves by subtracting the offset.
A microfluidic check valve and a method of using the check valve In microfluidic devices includes a check valve comprising two stacked chambers that are separated by a pore-containing membrane. The membrane is composed of an elastomeric material and can be configured in normally open or normally closed state. The normally open check valve can be implemented so that the degree of back pressure necessary to close the valve can be set.
F16K 1/00 - Lift valves, i.e. cut-off apparatus with closure members having at least a component of their opening and closing motion perpendicular to the closing faces
A microfluidic device adapted to perform many simultaneous binding assays including but not limited to immunological experiments, such as ELISA assays, with minimal cross-talk between primary and secondary antibodies.
The invention provides methods and devices for detecting, enumerating or identifying target nucleic acid molecules using immobilized capture probes and single molecule sequencing techniques.
An apparatus (100) for sequentially analyzing particles such as single cells or single beads, by spectrometry. The apparatus, an elemental flow cytometer, includes means (102) for sequential particle introduction, means (104) to vaporize, atomize and excite or ionize the particles, or an elemental tag associated with an analyte on the particles, and means (106) to analyze the elemental composition of the vaporized, atomized and excited or ionized particles, or an elemental tag associated with the particles. Methods for sequentially analyzing particles such as singe cells or single beads by spectrometry are also described.
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