ANTIGEN-COUPLED HYBRIDIZATION REAGENTS

The present disclosure provides high-performance hybridization reagents for use in a variety of hybridization assays and other related techniques. The hybridization reagents comprise an oligonucleotide probe and a bridging antigen, wherein the bridging antigen is recognized by a detectable antibody with high affinity. Also provided are compositions comprising panels of hybridization reagents specific for multiple different target nucleic acids and compositions comprising pairs of hybridization reagents and their complementary detectable antibodies. The paired hybridization reagents and detectable antibodies are useful in a variety of hybridization assays, particularly in highly multiplexed assays, where the structure of the bridging antigen is varied in tandem with variation in the detectable antibody, such that a multiplicity of hybridization reagents are provided that are capable of simultaneously detecting a multiplicity of target nucleic acids in a single assay. Also provided are kits ...

Methods of combining the detection of biomolecules into a single assay using fluorescent in situ sequencing

Simultaneous quantification of a plurality of proteins in a user-defined region of a cross-sectioned tissue

The present invention relates to, among other things, probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.

METHODS OF CLASSIFYING BIOLOGICAL SAMPLES FOR PREDICTING RESPONSE TO TYROSINE KINASE INHIBITOR TREATMENT

Gene copy numbers of signaling components downstream of EGFR identify non- small cell lung cancer (NSCLC) patients with poor outcomes on 2nd/3rd line gefitinib therapy.

METHOD FOR SPECIFIC, FAST DETECTION OF THREADLIKE BACTERIA

The invention relates to a method for specific, fast detection of threadlike bacteria, e.g. in activated sludge samples, by in situ-hybridisation. The invention also relates to oligonucoletide probes which are suitable for use in said method, in addition to kits which enable said detection method to be carried out.

チロシンキナーゼ阻害剤治療に対する反応を予測するために生体試料を分類する方法

Methods of combining the detection of biomolecules into a single assay using fluorescent in situ sequencing

The present disclosure provides methods that combine RNA fluorescent in situ sequencing (FISSEQ) with other molecular detection modalities, forming an integrated panomic detection platform. In various embodiments, the present disclosure provides systems and methods to prepare a biological sample to preserve the spatial relationships of biomolecules of interest within the biological sample for FISSEQ detection.

System and method for capturing multi-color fish images

Method of detection of DNA end(s) and its use

The invention concerns the method of detection of DNA end(s) in a biological material, comprising the following steps I – III and at least one of sub-steps a-h of each of steps I - III:

METHODS OF CLASSIFYING BIOLOGICAL SAMPLES FOR PREDICTING RESPONSE TO TYROSINE KINASE INHIBITOR TREATMENT

Gene copy numbers of signaling components downstream of EGFR identify non- small cell lung cancer (NSCLC) patients with poor outcomes on 2nd/3rd line gefitinib therapy.

분자 이미징 및 관련 방법

... 본 발명은 단일 분자의, 또는, 단일 분자들의 하나 이상의 컬렉션의, 이미징과, 이미징에 관련된 방법에 관한 것이다. 방법의 일 형태에서, 본 발명은 단일 분자 이미징 방법을 제공한다. 상기 방법은, a) 표적 분자에 특정하여 바인딩되는 제 1 부분과, 하나 이상 파장의 광과 상호작용하는 하나 이상의 화학기의 결과로 검출가능한 제 2 부분을 포함하는 프로브에 테스트 샘플을 노출시키는 단계 - 상기 프로브는 복합체 제공을 위해 표적 분자에 바인딩됨 - 와, b) 상기 하나 이상의 화학기와 상호작용하는 하나 이상 파장의 광에 상기 복합체를 노출시키는 단계와, c) 하나 이상의 단일 분자의 이미지 제공을 위해 상기 하나 이상의 화학기와 상호작용하는 하나 이상 파장의 광의 상호작용으로부터 결과를 검출하는 단계를 포함한다. 상기 이미지는 적어도 1 x 105 ㎛2 이상의 이미징 영역에 걸쳐 450nm보다 우수한 분해능을 갖고, 상기 이미지는 어떤 검출 설정 변화없이 단일 검출 단계에서 획득된다.

지수 래디언스로 테더링된 연결된 증폭

신호 증폭을 위해 지수 래디언스로 테더링된 연결된 증폭을 위한 조성물이 본원에 개시된다. 또한, 신호 증폭을 위해 지수 래디언스로 테더링된 연결된 증폭을 위한 키트가 본원에 개시된다. 또한, 신호 증폭을 위해 지수 래디언스로 테더링된 연결된 증폭을 위한 방법이 본원에 개시된다.

SIMULTANEOUS QUANTIFICATION OF A PLURALITY OF PROTEINS IN A USER-DEFINED REGION OF A CROSS-SECTIONED TISSUE

The present invention relates to, among other things, probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.

METHODS OF COMBINING THE DETECTION OF BIOMOLECULES INTO A SINGLE ASSAY USING FLUORESCENT IN SITU SEQUENCING

Simultaneous quantification of a plurality of proteins in a user-defined region of a cross-sectioned tissue

The present invention relates to, among other things, probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.

METHOD FOR SPECIFIC, FAST DETECTION OF THREADLIKE BACTERIA

The invention relates to a method for specific, fast detection of threadlike bacteria, e.g. in activated sludge samples, by in situ-hybridisation. The invention also relates to oligonucoletide probes which are suitable for use in said method, in addition to kits which enable said detection method to be carried out.

METHOD OF DETECTION OF DNA END(S) AND ITS USE

The invention concerns the method of detection of DNA end(s) in a biological material, comprising the following steps I III and at least one of sub-steps a-h of each of steps I - III: I. PREPARATION OF THE MATERIAL comprising a. fixation and/or permeabilization and/or lysis and/or isolation and/or fractionation and/or immobilization of the biological material, b. increasing accessibility of DNA end(s), c. blocking nonspecific binding site(s) for molecules type 2-6, in the biological material; II. PROCESSING OF DNA END(S) comprising d. ): modification of DNA end(s) by chemical or physical processing followed by binding molecules type 1 to the DNA end(s) by catalytic or noncatalytic means; blocking nonspecific binding site(s) for molecules type 2-6 in the biological material; III. RECOGNITION AND DETECTION OF THE MODIFIED DNA END(S): incubation of the biological material from step II with at least two molecules type 2 and 3 which bind to the molecules type 1 in a manner that allows steps ...

Compositions and methods of RNA analysis

The present disclosure relates to compositions and methods of RNA analysis. In particular, the present disclosure provides a method of RNA analysis that includes obtaining a sample, applying one or more multi-partite probes to the sample, where each of the one or more multi-partite probes includes at least two sub-probes, annealing at least one of the applied one or more multi-partite probes to at least one target nucleic acid within the sample, and ligating the at least two sub-probes associated with the at least one annealed multi-partite probe to create a target nucleic acid proxy that can be detected.

SYSTEM AND METHOD FOR CAPTURING MULTI-COLOR FISH IMAGES

A method of obtaining multi-color FISH images is provided. The method is effected by: (a) using a color imager for capturing a dark image of a magnified biological sample stained with as least two distinct FISH probes; (b) determining dark intensity (Id) for each pixel of the dark image; (c) using the color imager for capturing a fluorescently illuminated image of the magnified biological sample; and (d) correcting an intensity (I) of each pixel of the fluorescently illuminated image of the biological sample according to its dark intensity (Id) to thereby obtain a multi-color FISH image of the biological sample.

Antigen-coupled hybridization reagents

The present disclosure provides high-performance hybridization reagents for use in a variety of hybridization assays and other related techniques. The hybridization reagents comprise an oligonucleotide probe and a bridging antigen, wherein the bridging antigen is recognized by a detectable antibody with high affinity. Also provided are compositions comprising panels of hybridization reagents specific for multiple different target nucleic acids and compositions comprising pairs of hybridization reagents and their complementary detectable antibodies. The paired hybridization reagents and detectable antibodies are useful in a variety of hybridization assays, particularly in highly multiplexed assays, where the structure of the bridging antigen is varied in tandem with variation in the detectable antibody, such that a multiplicity of hybridization reagents are provided that are capable of simultaneously detecting a multiplicity of target nucleic acids in a single assay. Also provided are kits ...

METHODS OF CLASSIFYING BIOLOGICAL SAMPLES FOR PREDICTING RESPONSE TO TYROSINE KINASE INHIBITOR TREATMENT

Gene copy numbers of signaling components downstream of EGFR identify non- small cell lung cancer (NSCLC) patients with poor outcomes on 2nd/3rd line gefitinib therapy.

Analysis of chromatin using a nicking enzyme

Provided herein, among other things, are various compositions and methods for analyzing chromatin. In some embodiments, the composition may comprise a mixture of a nicking enzyme, four dNTPs, at least one labeled dNTP and, optionally, a polymerase. In some embodiments, this method may comprise: obtaining a sample comprising chromatin, reacting the sample with the composition to selectively label the open chromatin in the sample, and analyzing the labeled sample.

New oligonucleotides are useful to detect filamentous bacteria in samples, particularly in activated sludge

The invention relates to a method for specific, fast detection of threadlike bacteria, e.g. in activated sludge samples, by in situ-hybridisation. The invention also relates to oligonucoletide probes which are suitable for use in said method, in addition to kits which enable said detection method to be carried out.

Simultaneous quantification of a plurality of proteins in a user-defined region of a cross-sectioned tissue

The present invention relates to, among other things, probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.

MATRIX IMPRINTING AND CLEARING

핵산의 프로빙 및 맵핑을 위한 ＲＮＡ-가이드된 시스템

... 가이드 RNA 및 Cas9 단백질을 사용하여 표적 핵산을 검출, 프로빙, 맵핑 및 지정 서열분석하는 방법이 제공된다. 가이드 RNA/Cas9 복합체의 표적 핵산에의 결합을 검출하는 방법이며, 여기서 가이드 RNA는 프로브에 혼성화할 수 있는 3' 테일 서열을 포함하는 것인 방법이 제공된다. 가이드 RNA/Cas9 복합체의 표적 핵산에의 결합을 검출하는 방법이며, 여기서 복합체는 물리적으로 검출되는 것인 방법이 제공된다. [대표도] 도 6A ...

In situ hybridization detection reagent box of RhoGDI2 gene, detection method and use thereof

The invention relates to an in situ hybridization detection kit of a RhoGDI2 (Rho GDP dissociation inhibitor beta) gene, a detection method and an application thereof. The kit comprises a hybridization probe, a label, a hybridization fluid and a potentiating agent, wherein, the hybridization probe sequence is shown in SEQ ID N0.1. The in situ hybridization detection method of the RhoGDI2 gene comprises the following steps that: a. the hybridization probe in the kit is contacted with the RNA to be detected in a zymolyte to form a hybridization complex; and b. the hybridization complex obtained in the step a is detected. The invention also relates to the application of the kit in the preparation of drugs for detecting the early cancer metastasis and recurrent diseases. The kit has the characteristics of high sensitivity and strong specificity. Meanwhile, the detection method has simple and convenient operation and can be used and popularized in the hospital over the district level.

METHOD FOR SPECIFIC, FAST DETECTION OF THREADLIKE BACTERIA

The invention relates to a method for specific, fast detection of threadlike bacteria, e.g. in activated sludge samples, by in situ-hybridisation. The invention also relates to oligonucoletide probes which are suitable for use in said method, in addition to kits which enable said detection method to be carried out.

MATRIX IMPRINTING AND CLEARING

The present invention generally relates to systems and methods for imaging or determining nucleic acids or other desired targets, for instance, within cells or tissues. In one aspect, a sample is exposed to a plurality of nucleic acid probes that are determined within the sample. In some cases, however, background fluorescence or off-target binding may make it more difficult to determine properly bound nucleic acid probes. Accordingly, other components of the samples that may be contributing to the background, such as proteins, lipids, and/or other non-targets, may be “cleared” from the sample to improve determination. However, in certain embodiments, nucleic acids or other desired targets may be prevented from also being cleared, e.g., using polymers or gels within the sample. Other aspects are generally directed to compositions or kits involving such systems, methods of using such systems, or the like. 1. A method , comprising:exposing a sample to a plurality of nucleic acid probes;polymerizing a gel within the sample;anchoring a target to the gel;clearing non-targets from the sample; anddetermining the targets within the gel by determining binding of the nucleic acid probes by imaging.2. The method of claim 1 , wherein the target is a nucleic acid.3. The method of any one of or claim 1 , wherein the target comprises RNA.4. The method of any one of or claim 1 , wherein the target comprises DNA.5. The method of any one of - claim 1 , wherein anchoring the target to the gel comprises anchoring the target to a nucleic acid probe and covalently bonding the nucleic acid probe to the gel.6. The method of any one of - claim 1 , wherein anchoring the target to the gel comprises anchoring the target to a nucleic acid probe and noncovalently bonding the nucleic acid probe to the gel.7. The method of any one of - claim 1 , wherein anchoring the target to the gel comprises anchoring the target to the gel via hybridization to the nucleic acid probes.8. The method of any one of ...

SIMULTANEOUS QUANTIFICATION OF A PLURALITY OF PROTEINS IN A USER-DEFINED REGION OF A CROSS-SECTIONED TISSUE

The present invention relates to, among other things, probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.

COMPOSITIONS AND METHODS OF RNA ANALYSIS

The present disclosure relates to compositions and methods of RNA analysis. In particular, the present disclosure provides a method of RNA analysis that includes obtaining a sample, applying one or more multi-partite probes to the sample, where each of the one or more multi-partite probes includes at least two sub-probes, annealing at least one of the applied one or more multi-partite probes to at least one target nucleic acid within the sample, and ligating the at least two sub-probes associated with the at least one annealed multi-partite probe to create a target nucleic acid proxy that can be detected. 1. A method , comprising:obtaining a sample;applying one or more multi-partite probes to the sample, wherein each of the one or more multi-partite probes includes at least two sub-probes;annealing at least one of the applied one or more multi-partite probes to at least one target nucleic acid within the sample; andligating the at least two sub-probes associated with the at least one annealed multi-partite probe to create a target nucleic acid proxy that can be detected.2. The method of claim 1 , further comprising:releasing the target nucleic acid proxy from the target nucleic acid; andamplifying the target nucleic acid proxy.3. The method of claim 1 , wherein the at least one target nucleic acid is RNA.4. The method of claim 1 , wherein the sub-probes comprise appended primer binding sites to facilitate subsequent amplification of the target nucleic acid proxy.5. The method of claim 1 , wherein the at least two sub-probes are ligated with an enzyme claim 1 , a chemical reaction claim 1 , or a photoreaction.6. The method of claim 5 , wherein the enzyme is a ligase.7. The method of claim 6 , wherein the ligase is selected from the group consisting of a T4 RNA Ligase 2 (Rnl2) claim 6 , T4 DNA ligase claim 6 , a Chlorella virus DNA Ligase (PBCV-1 DNA Ligase) claim 6 , a Rnl2 derivative claim 6 , PBCV-1 derivative claim 6 , and any combination thereof.8. The method of ...

Analysis of chromatin using a nicking enzyme

Provided herein, among other things, are various compositions and methods for analyzing chromatin. In some embodiments, the composition may comprise a mixture of a nicking enzyme, four dNTPs, at least one labeled dNTP and, optionally, a polymerase. In some embodiments, this method may comprise: obtaining a sample comprising chromatin, reacting the sample with the composition to selectively label the open chromatin in the sample, and analyzing the labeled sample.

METHODS AND DEVICES FOR DETECTION AND ACQUISITION OF BIOMARKERS

... 본 발명은 피험체 제자리(insitu)로부터 분자 바이오마커를 검출하고 캡쳐하기 위한 디바이스 및 방법을 제공한다. 구체적으로, 디바이스는 관심의 하나 이상의 바이오마커에 특이적인 프로브에 부착된 마이크로니들 어레이를 포함한다. 디바이스는 피험체의 신체(예를 들어, 조직, 혈류) 내 바이오마커를 검출하는 데 있어 피험체에 직접(예를 들어, 피부 관통을 통해) 사용될 수 있다.

SIMULTANEOUS QUANTIFICATION OF A PLURALITY OF PROTEINS IN A USER-DEFINED REGION OF A CROSS-SECTIONED TISSUE

절편화된 조직의 사용자-한정된 영역에서의 복수의 단백질의 동시적인 정량화

... 본 발명은 특히, 사용자-한정된(user-defined) 조직 영역, 사용자-한정된 세포, 및/또는 세포에서 사용자-한정된 세포내 구조에서 단백질 발현의 동시적인, 다중화된(multiplexed) 검출 및 정량화를 위한 프로브, 조성물, 방법 및 키트에 관한 것이다.

Using repetitive sequences for DNA quality assessments

The present disclosure provides methods, arrays and kits for assessing the quality of genomic DNA samples, especially those obtained from formalin-fixed paraffin-embedded (FFPE) samples. The methods, arrays and kits provided herein use primer pairs specific to regions in the genomes of the organisms from which genomic DNA samples are obtained that have identical or nearly identical copies distributed across multiple chromosomes.

SYSTEM AND METHOD FOR CAPTURING MULTI-COLOR FISH IMAGES

A method of obtaining multi-color FISH images is provided. The method is effected by: (a) using a color imager for capturing a dark image of a magnified biological sample stained with as least two distinct FISH probes; (b) determining dark intensity (Id) for each pixel of the dark image; (c) using the color imager for capturing a fluorescently illuminated image of the magnified biological sample; and (d) correcting an intensity (I) of each pixel of the fluorescently illuminated image of the biological sample according to its dark intensity (Id) to thereby obtain a multi-color FISH image of the biological sample.

METHOD OF DETECTION OF DNA END(S) AND ITS USE

The invention concerns the method of detection of DNA end(s) in a biological material, comprising the following steps I III and at least one of sub-steps a-h of each of steps I-III: I. PREPARATION OF THE MATERIAL comprising a fixation and/or permeabilization and/or lysis and/or isolation and/or fractionation and/or immobilization of the biological material, b. increasing accessibility of DNA end(s), c. blocking nonspecific binding site(s) for molecules type 2-6, in the biological material; H. PROCESSING OF DNA END(S) comprising d.): modification of DNA end(s) by chemical or physical processing followed by binding molecules type 1 to the DNA end(s) by catalytic or noncatalytic means; blocking nonspecific binding site(s) for molecules type 2-6 in the biological material; III RECOGNITION AND DETECTION OF THE MODIFIED DNA END(S): incubation of the biological material from step II with at least two molecules type 2 and 3 which bind to the molecules type 1 in a manner that allows steps leading to rolling circle amplification (RCA) reactions, g. detection of DNA end(s) by: i. optionally contacting suitable molecules type 4 and/or 5 with molecules type 2 and 3, wherein the molecules type 4 and/or 5 are conjugated with the oligonucleotides type 1, ii. adding oligonucleotides type 2 and enzyme ligase to allow hybridization of said added oligonucleotides type 2 to the oligonucleotides type 1 already linked to molecules type 4 and/or 5, or to molecules type 2 and 3 if they are linked to oligonucleotides type 1, and subsequently performing DNA ligation of oligonucleotides type 2, iii. performing amplification by adding enzyme polymerase and a solution of nucleotides to allow rolling circle amplification (RCA) reactions, and molecules type 6 to allow subsequent hybridization of molecules type 6 to thus obtained product of RCA reactions, h. detection of molecules type 6; wherein when more than one sub-step a-c of step I is performed then they may occur in any order. The invention ...

Multiplex genome engineering in eukaryotes

Compositions and methods for gene editing are provided. The methods employ an oligo-based annealing mechanism that is rooted in the process of DNA replication rather than homologous recombination (HR). Oligo incorporation efficiencies are comparable and often exceed those of CRISPR/cas9 editing without the need for double strand breaks (DSBs). By relying on the multiplex annealing of oligos rather than DSBs the process is highly scalable across a genomic region of interest and can generate many scarless modifications of a chromosome simultaneously. Combinatorial genomic diversity can be generated across a population of cells in a single transformation event; genomic landscapes can be traversed through successive iterations of the process, and genome-wide changes can be massively parallelized and amplified through systematic strain mating.

METHODS TO FURTHER ENHANCE SIGNAL AMPLIFICATION FOR THE IN SITU DETECTION OF NUCLEIC ACIDS

The present invention relates to detection of nucleic acids and provides a composition comprising a Signal Generating Complex, wherein the composition comprises: (A) a pair of target probes (TPs), wherein a first TP of the pair of TPs comprises a nucleic acid sequence comprising two segments; (B) a pair of base PPAs comprising the first and second base PPAs, wherein the first base PPA comprises a nucleic acid sequence comprising three segments; (C) a set of extension PPAs comprising the first and second extension PPAs, wherein the first extension PPA comprises a nucleic acid sequence comprising two segments; (D) a plurality of pre-amplifiers (PAs), wherein the PAs comprise a nucleic acid sequence comprising three segments; (E) a plurality of amplifiers (AMPs), wherein the AMPs comprise a nucleic acid sequence comprising two segments; and (F) a plurality of label probes (LPs), wherein the LPs comprise a nucleic acid sequence comprising two segments. 1. A composition comprising a Signal Generating Complex (SGC) , wherein the composition comprises:(A) a pair of target probes (TPs), wherein a first TP of the pair of TPs comprises a nucleic acid sequence comprising two segments, (i) a segment comprising a binding site for a first segment of a target nucleic acid, and (ii) a segment comprising a binding site for a first base pre-pre-amplifier (base PPA); and wherein a second TP of the pair of TPs comprises a nucleic acid sequence comprising two segments, (i) a segment comprising a binding site for a second segment of the target nucleic acid, and (ii) a segment comprising a binding site for a second base PPA;(B) a pair of base PPAs comprising the first and second base PPAs, wherein the first base PPA comprises a nucleic acid sequence comprising three segments, (i) a segment that binds to the first base PPA binding site of the first TP, (ii) a segment comprising a plurality of first pre-amplifier binding segment repeats (first PA-BSRs), and (iii) a segment comprising a ...

BRANCHED PROXIMITY HYBRIDIZATION ASSAY

The invention relates to a method for detecting the proximity of at least two biomolecules using branched DNA technology. The assay is called branched proximity hybridization assay. 1. Method for detecting the proximity of at least two target biomolecules comprising:Providing at least two target biomolecules,providing at least two Target Binding Reagents, each binding to at least one of said target biomolecules,wherein at least one designed Oligo Extension, comprising a linker and a complementary sequence to a Z-DNA Probe or a complementary sequence to a Pre-amplifier, is attached to each Target Binding Reagent,binding of at least two Target Binding Reagents with different Oligo Extensions to the at least two target biomolecules andhybridization of two Z-DNA Probes, that are in vicinity, to Pre-amplifiers, or hybridization of the sequence complementary to a Pre-amplifier to Pre-amplifiers,forming a bDNA structure, wherein said bDNA structure comprises Pre-amplifiers and Amplifiershybridization of Label Probes anddetection.2. Method according to claim 1 , wherein the Target Binding Reagent is a biomolecule claim 1 , preferred selected from the group comprising nucleic acid sequences claim 1 , aptamers claim 1 , antibodies claim 1 , Fab claim 1 , nanobodies and scFv.3. Method according to or claim 1 , wherein said two Z-DNA Probes bind to the complementary sequences of the Oligo Extensions.4. Method according to at least one of the preceding claims claim 1 , wherein the molar ratio between Target Binding Reagent and Oligo Extension is 1:1.5. Method according to at least one of the preceding claims claim 1 , wherein the method is a high-throughput method.6. Method according to at least one of the preceding claims claim 1 , wherein the detection is a fluorescent or an enzymatic detection.7. Method according to at least one of the preceding claims claim 1 , wherein the method is a multiplex assay.8. Method according to at least one of the preceding claims claim 1 , ...

DNA quality assessments using repetitive sequences

The present disclosure provides methods, arrays and kits for assessing the quality of genomic DNA samples, especially those obtained from formalin-fixed paraffin-embedded (FFPE) samples. The methods, arrays and kits provided herein use primer pairs specific to regions in the genomes of the organisms from which genomic DNA samples are obtained that have identical or nearly identical copies distributed across multiple chromosomes.

Methods Of Classifying Biological Samples For Predicting Response To Tyrosine Kinase Inhibitor Treatment

Gene copy numbers of signaling components downstream of EGFR identify non-small cell lung cancer (NSCLC) patients with poor outcomes on 2nd/3rd line gefitinib therapy.

Methods of Hybridizing Probes to Genomic DNA

The present invention relates to methods of hybridizing nucleic acid probes to genomic DNA. 1. A method of improving binding efficiency of a labeled probe to double stranded DNA having a portion of the double stranded DNA separated into a first single strand segment and a complementary single strand segment comprisingcombining the double stranded DNA with a labeled probe that is complementary to the first single strand segment at a target sequence and one or more anti-lock probes that are complementary to either the first single strand segment or the complementary single strand segment wherein the labeled probe binds to the first single strand segment at the target sequence and the one or more anti-lock probes bind to at least the complementary single strand segment.2. The method of wherein the double stranded DNA is genomic DNA.3. The method of wherein the bound one or more anti-lock probes inhibits re-annealing of the first single strand segment and the complementary single strand segment.4. The method of wherein the labeled probe is between 2 nucleotides and 200 nucleotides in length.5. The method of wherein the labeled probe is an oligonucleotide paint.6. The method of wherein a first anti-lock probe binds to the complementary single strand segment at a position which overlaps with the bound labeled probe.7. The method of wherein a first anti-lock probe binds to the complementary single strand segment at a position which overlaps with the region complementary to the target sequence of the bound labeled probe.8. The method of wherein one or more anti-lock probes bind to the complementary single stranded segment at a position neighboring the region complementary to the target sequence of the bound labeled probe without overlap.9. The method of wherein a first anti-lock probe binds to the complementary single stranded segment at a position which overlaps with the region complementary to the target sequence of the bound labeled probe by at least one nucleotide.10. ...

METHODS OF CLASSIFYING BIOLOGICAL SAMPLES FOR PREDICTING RESPONSE TO TYROSINE KINASE INHIBITOR TREATMENT

METHODS FOR PERFORMING SPATIAL PROFILING OF BIOLOGICAL MOLECULES

The present disclosure provides methods, devices and systems that enable determination of spatial information of biological molecules by reacting the biological molecules with a zipcode array. In some examples, the zipcode array may code for the spatial positions of biological molecules attached to distinct positions on the zipcode array. In some examples, the spatial positions are 2-dimensional. In some cases, the spatial positions are 3-dimensional. In some examples, the present disclosure provides methods to detect spatial gene expression. In some examples, the present disclosure provides methods to detect spatial distribution of proteins. 1100-. (canceled)101. A method for detecting spatial distribution of a plurality of target molecules within a biological sample , comprisinga) contacting an oligonucleotide hydrogel with a spatial barcode array, wherein the oligonucleotide hydrogel comprises a plurality of immobilized complementary deoxyribonucleic acid (cDNA) molecules, wherein the spatial barcode array comprises a plurality of oligonucleotides, wherein each member of the plurality of oligonucleotides comprises a barcode sequence that identifies a location of the member of the plurality of oligonucleotides on the spatial barcode array, wherein the barcode sequence is indicative of the location of the member of the plurality of oligonucleotides on the spatial barcode array to within 2 μm;b) extending a first immobilized cDNA molecule of the plurality of immobilized cDNA molecules to a first tagged cDNA molecule of a plurality of tagged cDNA molecules, wherein the first tagged cDNA molecule comprises the first immobilized cDNA molecule and a complimentary copy of a first barcode sequence identifying a first location on the spatial barcode array;c) amplifying the plurality of tagged cDNA molecules to provide a plurality of amplified tagged cDNA molecules;d) sequencing at least a portion of the plurality of amplified tagged cDNA molecules and determining the ...

COMPOSITIONS AND METHODS OF RNA ANALYSIS

The present disclosure relates to compositions and methods of RNA analysis. In particular, the present disclosure provides a method of RNA analysis that includes obtaining a sample, applying one or more multi-partite probes to the sample, where each of the one or more multi-partite probes includes at least two sub-probes, annealing at least one of the applied one or more multi-partite probes to at least one target nucleic acid within the sample, and ligating the at least two sub-probes associated with the at least one annealed multi-partite probe to create a target nucleic acid proxy that can be detected.

SIMULTANEOUS QUANTIFICATION OF A PLURALITY OF PROTEINS IN A USER-DEFINED REGION OF A CROSS-SECTIONED TISSUE

The present invention relates to, among other things, probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.

Methods for the Imaging of Nucleic Acid Sequences

The present invention relates to methods of providing sequence specificity to in situ genome imaging at the level of the electron microscope (EM).

METHOD FOR SPECIFIC, FAST DETECTION OF THREADLIKE BACTERIA

The invention relates to a method for specific, fast detection of threadlike bacteria, e.g. in activated sludge samples, by in situ-hybridisation. The invention also relates to oligonucoletide probes which are suitable for use in said method, in addition to kits which enable said detection method to be carried out.

Simultaneous quantification of a plurality of proteins in a user-defined region of a cross-sectioned tissue

The present invention relates to, among other things, probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.

Analysis of Chromatin Using a Nicking Enzyme

Provided herein, among other things, are various compositions and methods for analyzing chromatin. In some embodiments, the composition may comprise a mixture of a nicking enzyme, four dNTPs, at least one labeled dNTP and, optionally, a polymerase. In some embodiments, this method may comprise: obtaining a sample comprising chromatin, reacting the sample with the composition to selectively label the open chromatin in the sample, and analyzing the labeled sample. 142.-. (canceled)43. A composition comprising a mixture of a nicking enzyme , four dNTPs , and at least one labeled dNTP.44. The composition of claim 43 , wherein the nicking enzyme is methylation-insensitive or methylation-dependent.45. The composition according to claim 44 , wherein the nicking enzyme is dependent on C or A in the recognition sequence for the nicking enzyme.46. The composition according to claim 43 , wherein the mixture is contained in or on a reaction vessel claim 43 , a microscope slide or a microtiter plate.47. The composition according to claim 43 , wherein the mixture comprises a non-naturally occurring buffering agent or is lyophilized.48. The composition according to claim 43 , wherein the composition further comprises chromatin.49. The composition of claim 48 , wherein the chromatin comprises open chromatin and closed chromatin claim 48 , and wherein the open chromatin is selectively labeled using a methylation independent claim 48 , a methylation sensitive nicking enzyme or a methylation dependent nicking enzyme.50. The composition according to claim 43 , wherein the composition further comprises permeabilized nuclei that contains the chromatin.51. The composition according to claim 48 , wherein the chromatin is from a clinical sample.52. The composition of claim 51 , wherein the clinical sample is a tumor biopsy.53. The composition of claim 44 , wherein the nicking enzyme is Nt. CviPII.54. The composition according to claim 43 , wherein at least one of the at least one labeled ...

Multiplexed single molecule RNA visualization with a two-probe proximity ligation system

SNAIL provides cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids, e.g. at least one, to up to 5, up to 10, up to 15, up to 20 or more transcripts can be simultaneously analyzed, at a rate of up to about 50, 100, 250, 500 or more cells/second. The methods require only two primers for amplification, and may further include a detection primer.

Method for the specific fast detection of threadlike bacteria

The invention relates to a method for specific, fast detection of threadlike bacteria, e.g., in activated sludge samples, by in situ hybridization. The invention also relates to oligonucleotide probes which are suitable for use in said method, in addition to kits which enable said detection method to be carried out.

MULTIPLEXED SINGLE MOLECULE RNA VISUALIZATION WITH TWO-PROBE PROXIMITY LIGATION SYSTEM

In situ hybridization detection reagent box of RhoGDI2 gene, detection method and use thereof

The invention relates to an in situ hybridization detection kit of a RhoGDI2 (Rho GDP dissociation inhibitor beta) gene, a detection method and an application thereof. The kit comprises a hybridization probe, a label, a hybridization fluid and a potentiating agent, wherein, the hybridization probe sequence is shown in SEQ ID N0.1. The in situ hybridization detection method of the RhoGDI2 gene comprises the following steps that: a. the hybridization probe in the kit is contacted with the RNA to be detected in a zymolyte to form a hybridization complex; and b. the hybridization complex obtainedin the step a is detected. The invention also relates to the application of the kit in the preparation of drugs for detecting the early cancer metastasis and recurrent diseases. The kit has the characteristics of high sensitivity and strong specificity. Meanwhile, the detection method has simple and convenient operation and can be used and popularized in the hospital over the district level.

Methods to further enhance signal amplification for the in situ detection of nucleic acids

The present invention relates to detection of nucleic acids and provides a composition comprising a Signal Generating Complex, wherein the composition comprises: (A) a pair of target probes (TPs), wherein a first TP of the pair of TPs comprises a nucleic acid sequence comprising two segments; (B) a pair of base PPAs comprising the first and second base PPAs, wherein the first base PPA comprises a nucleic acid sequence comprising three segments; (C) a set of extension PPAs comprising the first and second extension PPAs, wherein the first extension PPA comprises a nucleic acid sequence comprising two segments; (D) a plurality of pre-amplifiers (PAs), wherein the PAs comprise a nucleic acid sequence comprising three segments; (E) a plurality of amplifiers (AMPs), wherein the AMPs comprise a nucleic acid sequence comprising two segments; and (F) a plurality of label probes (LPs), wherein the LPs comprise a nucleic acid sequence comprising two segments.

DNA QUALITY CONTROLS

The present disclosure provides methods, arrays and kits for assessing the quality of genomic DNA samples, especially those obtained from formalin-fixed paraffin-embedded (FFPE) samples. The methods, arrays and kits provided herein use primer pairs specific to regions in the genomes of the organisms from which genomic DNA samples are obtained that have identical or nearly identical copies distributed across multiple chromosomes. 1. A method for assessing the quality of a test genomic DNA sample for downstream molecular DNA analyses , comprising:(a) performing one or more real-time PCR reactions that use genomic DNA in a test genomic DNA sample as templates in the presence of two or more primer pairs, wherein each of the two or more primer pairs is specific for amplifying identical or nearly identical genomic DNA fragments that are present at 10 or more different locations in the genome of the organism from which the test genomic DNA sample is obtained, wherein a genomic DNA fragment is nearly identical to another DNA if (i) the size difference between the two genomic DNA fragments is at most 5% of the full length of the longer fragment, and (ii) the sequence identity between the two fragments is at least 95%,(b) performing one or more real-time PCR reactions that use genomic DNA in a control genomic DNA sample as templates in the presence of the two or more primer pairs used in step (a),(c) determining the Ct values for the one or more real-time PCR reactions in step (a),(d) determining the Ct values for the one or more real-time PCR reactions in step (b),(e) determining the difference (ΔCt) between the Ct values determined in step (c) and the corresponding Ct values determined in step (d) for the one or more real-time PCR reactions, wherein the difference is indicative of the quality of the test genomic DNA sample, and(f) performing one or more additional molecular analyses of the genomic DNA test sample whose quality has been assessed as suitable for such analysis ...

DNA QUALITY CONTROLS

The present disclosure provides methods, arrays and kits for assessing the quality of genomic DNA samples, especially those obtained from formalin-fixed paraffin-embedded (FFPE) samples. The methods, arrays and kits provided herein use primer pairs specific to regions in the genomes of the organisms from which genomic DNA samples are obtained that have identical or nearly identical copies distributed across multiple chromosomes. 1. A method for assessing the quality of a test genomic DNA sample for a downstream molecular DNA analysis , comprising:(a) performing one or more real-time PCR reactions that use genomic DNA in a test genomic DNA sample as templates in the presence of two or more primer pairs, wherein each of the two or more primer pairs is specific for amplifying identical or nearly identical genomic DNA fragments that are present at 10 or more different locations in the genome of the organism from which the test genomic DNA sample is obtained, wherein a genomic DNA fragment is nearly identical to another DNA if (i) the size difference between the two genomic DNA fragments is at most 5% of the full length of the longer fragment, and (ii) the sequence identity between the two fragments is at least 95%,(b) determining the Ct values for the one or more real-time PCR reactions in step (a),(c) determining the difference (ΔCt) between the Ct values determined in step (b) and the corresponding control Ct values of one or more real-time PCR reactions using genomic DNA in a control genomic DNA sample as templates in the presence of the two or more primer pairs used in step (a), wherein the difference is indicative of the quality of the test genomic DNA sample, and(d) performing one or more additional molecular analyses of the genomic DNA test sample whose quality has been assessed as suitable for such analysis.2. The method of claim 1 , wherein the method comprises:(i) measuring PCR amplifiable molecules in the test genomic DNA sample using the difference in the Ct ...

Methods of Combining the Detection of Biomolecules Into a Single Assay Using Fluorescent In Situ Sequencing

The present disclosure provides methods that combine RNA fluorescent in situ sequencing (FISSEQ) with other molecular detection modalities, forming an integrated panomic detection platform. In various embodiments, the present disclosure provides systems and methods to prepare a biological sample to preserve the spatial relationships of biomolecules of interest within the biological sample for FISSEQ detection.

Method for the specific fast detection of threadlike bacteria

The invention relates to a method for specific, fast detection of threadlike bacteria, e.g., in activated sludge samples, by in situ hybridization. The invention also relates to oligonucleotide probes which are suitable for use in said method, in addition to kits which enable said detection method to be carried out.

Molecular imaging and related methods

The present invention generally relates to imaging single molecules, or one or more collections of single molecules, and methods related to the imaging. In a method aspect, the present invention provides a method of imaging single molecules. The method comprises the steps of: a) exposing a test sample to a probe, wherein the probe comprises a first portion that specifically binds to a target molecule and a second portion that is detectable as the result of one or more chemical groups that interact with light at one or more wavelengths, wherein the probe binds to a target molecule to provide a complex; b) exposing the complex to one or more wavelengths of light that interact with the one or more chemical groups; c) detecting a result from the interacting of one or more wavelengths of light that interact with the one or more chemical groups to provide an image of one or more single molecules. The image possesses a resolution better than 450 nm over a view field area of at least 1×10 5 μm 2 , and wherein the image is obtained in a single detection step without variation of any detection settings.

ANTIGEN-COUPLED HYBRIDIZATION REAGENTS

The present disclosure provides high-performance hybridization reagents for use in a variety of hybridization assays and other related techniques. The hybridization reagents comprise an oligonucleotide probe and a bridging antigen, wherein the bridging antigen is recognized by a detectable antibody with high affinity. Also provided are compositions comprising panels of hybridization reagents specific for multiple different target nucleic acids and compositions comprising pairs of hybridization reagents and their complementary detectable antibodies. The paired hybridization reagents and detectable antibodies are useful in a variety of hybridization assays, particularly in highly multiplexed assays, where the structure of the bridging antigen is varied in tandem with variation in the detectable antibody, such that a multiplicity of hybridization reagents are provided that are capable of simultaneously detecting a multiplicity of target nucleic acids in a single assay. Also provided are kits comprising the hybridization reagents, methods of hybridization assay using the hybridization reagents of the disclosure, and methods of preparation of the hybridization reagents. 2. The hybridization reagent composition of claim 1 , wherein the bridging antigen is a peptide.3. The hybridization reagent composition of claim 1 , wherein the bridging antigen comprises a plurality of antigenic determinants.4. The hybridization reagent composition of claim 3 , wherein each antigenic determinant in the plurality of antigenic determinants is the same.5. The hybridization reagent composition of claim 3 , wherein the plurality of antigenic determinants comprises a linear repeating structure.6. The hybridization reagent composition of claim 5 , wherein the linear repeating structure is a linear repeating peptide structure.7. The hybridization reagent composition of claim 3 , wherein the plurality of antigenic determinants comprises at least three antigenic determinants.8. The hybridization ...

MULTIPLEXED SINGLE MOLECULE RNA VISUALIZATION WITH A TWO-PROBE PROXIMITY LIGATION SYSTEM

SNAIL provides cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids, e.g. at least one, to up to 5, up to 10, up to 15, up to 20 or more transcripts can be simultaneously analyzed, at a rate of up to about 50, 100, 250, 500 or more cells/second. The methods require only two primers for amplification, and may further include a detection primer. 1. A method for determining the abundance of a target nucleic acid in a single cell , the method comprising:contacting a fixed and permeabilized cell with at least one pair of SNAIL oligonucleotide primers under conditions permissive for specific hybridization, wherein the pair of primers comprises a Splint Primer Oligonucleotide (SPO) and a Padlock Oligonucleotide (PO), wherein each of SPO and PO comprise a first complementarity region (CR1 and CR1′, respectively) complementary to adjacent sequences on the target nucleic acid; and each of SPO and PO further comprise a second complementarity region (CR2 and CR2′) located adjacent to CR1 or CR1′; wherein CR2′ is a split region such that the 5′ and the 3′ ends of PO hybridize to CR2 such that after hybridization the 5′ and the 3′ ends of PO are positioned directly adjacent to one another;washing the cell free of unbound primer;contacting the cell with ligase wherein the PO is ligated to generate a closed circle;performing rolling circle amplification using the PO as a template and SPO as a primer for a polymerase;contacting the cell with a detection probe under conditions permissive for specific hybridization; anddetecting the level of bound detection probes to determine the abundance of the target nucleic acid.2. The method of claim 1 , wherein the SNAIL oligonucleotide primer pairs are denatured by heating before contacting the sample.3. The method of claim 1 , wherein the cell is present in a population of cells.4. The method of claim 3 , ...

Analysis of Chromatin Using a Nicking Enzyme

Provided herein, among other things, are various compositions and methods for analyzing chromatin. In some embodiments, the composition may comprise a mixture of a nicking enzyme, four dNTPs, at least one labeled dNTP and, optionally, a polymerase. In some embodiments, this method may comprise: obtaining a sample comprising chromatin, reacting the sample with the composition to selectively label the open chromatin in the sample, and analyzing the labeled sample. 142.-. (canceled)43. A method for detecting abnormal cells in or from a tissue section or biopsy wherein the abnormal cells have altered chromatin compared to normal cells , comprising:(a) reacting the cells with a composition comprising a mixture of a nicking enzyme, four dNTPs, and at least one labeled dNTP, and a polymerase to selectively label the chromatin;(b) incorporating the labeled dNTP into the chromatin of the cells to form labelled nucleic acid; and(c) analyzing the pattern of labelled dNMPs in the labeled cells.44. The method according to claim 43 , wherein (c) further comprises determining whether the pattern of labelled dNMPs correspond to a cancer diagnosis of the cells or tissues.45. The method according to claim 43 , wherein prior to (a) claim 43 , making the cells permeable.46. The method according to claim 43 , wherein the tissue section or biopsy is fixed.47. The method according to claim 45 , wherein the tissue section or biopsy is fixed.48. The method according to claim 43 , wherein after (b) reverse cross linking and isolating DNA from the cells.49. The method according to claim 43 , wherein following (b) claim 43 , labelling nuclei from the cells with a primary antibody.50. The method according to claim 49 , wherein after labelling with a primary antibody claim 49 , labelling with a secondary antibody.51. The method according to claim 49 , further comprising: visualizing the labelled nuclei using a fluorescent microscope. The mammalian genome is largely packaged into chromatin ...

MULTIPLEXED SINGLE MOLECULE RNA VISUALIZATION WITH A TWO-PROBE PROXIMITY LIGATION SYSTEM

SNAIL provides cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids, e.g. at least one, to up to 5, up to 10, up to 15, up to 20 or more transcripts can be simultaneously analyzed, at a rate of up to about 50, 100, 250, 500 or more cells/second. The methods require only two primers for amplification, and may further include a detection primer. 1. A method for determining the abundance of a target nucleic acid in a single cell , the method comprising:contacting a fixed and permeabilized cell with at least one pair of SNAIL oligonucleotide primers under conditions permissive for specific hybridization, wherein the pair of primers comprises a Splint Primer Oligonucleotide (SPO) and a Padlock Oligonucleotide (PO), wherein each of SPO and PO comprise a first complementarity region (CR1 and CR1′, respectively) complementary to adjacent sequences on the target nucleic acid; and each of SPO and PO further comprise a second complementarity region (CR2 and CR2′) located adjacent to CR1 or CR1′; wherein CR2′ is a split region such that the 5′ and the 3′ ends of PO hybridize to CR2 such that after hybridization the 5′ and the 3′ ends of PO are positioned directly adjacent to one another;washing the cell free of unbound primer;contacting the cell with ligase wherein the PO is ligated to generate a closed circle;performing rolling circle amplification using the PO as a template and SPO as a primer for a polymerase;contacting the cell with a detection probe under conditions permissive for specific hybridization; anddetecting the level of bound detection probes to determine the abundance of the target nucleic acid.2. The method of claim 1 , wherein the SNAIL oligonucleotide primer pairs are denatured by heating before contacting the sample.3. The method of claim 1 , wherein the cell is present in a population of cells.4. The method of claim 3 , ...

DNA quality assessments using repetitive sequences

The present disclosure provides methods, arrays and kits for assessing the quality of genomic DNA samples, especially those obtained from formalin-fixed paraffin-embedded (FFPE) samples. The methods, arrays and kits provided herein use primer pairs specific to regions in the genomes of the organisms from which genomic DNA samples are obtained that have identical or nearly identical copies distributed across multiple chromosomes.

DNA QUALITY CONTROLS

The present disclosure provides methods, arrays and kits for assessing the quality of genomic DNA samples, especially those obtained from formalin-fixed paraffin-embedded (FFPE) samples. The methods, arrays and kits provided herein use primer pairs specific to regions in the genomes of the organisms from which genomic DNA samples are obtained that have identical or nearly identical copies distributed across multiple chromosomes.

MOLECULAR IMAGING AND RELATED METHODS

A method of imaging single molecules includes exposing a test sample to a probe. The probe includes a first portion that specifically binds to a target molecule and a second portion that is detectable as the result of one or more chemical groups that interact with light at one or more wavelengths. The probe binds to a target molecule to provide a complex. The method also includes exposing the complex to one or more wavelengths of light that interact with the one or more chemical groups; and detecting a result from the interaction of the light and the one or more chemical groups to provide an image of the one or more single molecules. The image possesses a resolution better than 450 nm over a view field area of at least 1×105 μm2, and the image is obtained in a single detection step without variation of any detection settings.

ANTIGEN-COUPLED HYBRIDIZATION REAGENTS

Methods of Polynucleotide Detection

The present invention provides methods of detecting for the presence of a polynucleotide in vivo. These methods are particularly useful for performing identification and/or analysis of samples or specimens in which it is impossible, impractical, or undesirable to move or remove them from their current environment. Methods of practicing the present invention for the purpose of identifying and/or analyzing transgenic plant tissue or cells, in addition to animal tissue or cells and bacterial cells are also provided.

METHOD AND PRODUCT FOR LOCALIZED OR SPATIAL DETECTION OF NUCLEIC ACID IN A TISSUE SAMPLE

Localized detection of RNA in a tissue sample that includes cells is accomplished on an array. The array include a number of features on a substrate. Each feature includes a different capture probe immobilized such that the capture probe has a free 3′ end. Each feature occupies a distinct position on the array and has an area of less than about 1 mm. Each capture probe is a nucleic acid molecule, which includes a positional domain including a nucleotide sequence unique to a particular feature, and a capture domain including a nucleotide sequence complementary to the RNA to be detected. The capture domain can be at a position 3′ of the positional domain. 1. A method for localized detection of RNA in a tissue sample comprising cells , said method comprising:{'sup': '2', '(a) providing an array comprising a plurality of features on a substrate, each feature comprising a different capture probe immobilized thereon such that the capture probe has a free 3′ end, each feature occupying a distinct position on the array and having an area of less than about 1 mm, each capture probe consisting of a nucleic acid molecule comprising the following domains oriented 5′ to 3′(i) a positional domain comprising a nucleotide sequence unique to a particular feature; and(ii) a capture domain comprising a nucleotide sequence complementary to the RNA to be detected;(b) contacting said array with the tissue sample comprising cells such that the tissue sample contacts a plurality of the features at their distinct positions on the array;(c) hybridizing the RNA present in the tissue sample comprising cells that are complementary to the capture sequences of the capture probes immobilized on the features, such that the RNA is captured by the capture domain of the capture probes in the features;(d) generating cDNA molecules from the captured RNA, by extending the capture probes enzymatically using the captured RNA as an extension template, such that the cDNA molecules comprise the nucleotide ...

Method of Preparing Cell Free Nucleic Acid Molecules by In Situ Amplification

Methods for in situ amplification (ISA) of cfNA, such as cfDNA, in a sample are provided wherein the cfNA in the sample is not subject to a nucleic acid purification step. The methods disclosed may be used to generate an analyzable pool of cfNA present in the sample. The analyzable pool may be used with a variety of analytical techniques to characterize the nucleic acid in the sample. Methods of diagnosis, determining a therapeutic intervention and monitoring of a subject are also provided. 1. A method of in situ amplification of a cell-free nucleic acid (cfNA) the method comprising the steps of:a. providing a liquid sample containing a plurality of cfNA;b. performing at least one processing step on the sample;c. subjecting the sample to a sequential heating program and converting at least a portion of the cfNA in the sample to a modified cfNA using an enzyme mixture to add an exogenous nucleic acid sequence to at least one of the 5′ or 3′ ends of at least a portion of the cfNA in the sample to create an amplifiable cfNA pool, wherein the exogenous nucleic acid sequence contains a primer site capable of binding a primer and the exogenous nucleic acid sequence has a degenerate nucleic acid sequence flanking at least one side of the primer site; andd. amplifying the amplifiable cfNA pool to produce an analyzable pool of cfNA.wherein the cfNA in the sample is not subject to a nucleic acid purification step.2. The method of claim 1 , wherein the cfNA is selected from the group consisting of: cfDNA and cfRNA.3. (canceled)4. The method of claim 1 , wherein the cfNA is cfRNA and the cfRNA is converted to double-strand DNA prior to step (c).5. (canceled)6. The method of claim 1 , wherein at least a portion of the cfNA in the sample are ligated together.7. The method of claim 1 , wherein the cfNA in the sample has a fragment size distribution of 50 bp to 2 claim 1 ,000 bp claim 1 , 100 bp to 1 claim 1 ,000 bp claim 1 , 50 bp to 600 bp claim 1 , 100 bp to 500 bp claim 1 , 100 ...

METHOD AND PRODUCT FOR LOCALIZED OR SPATIAL DETECTION OF NUCLEIC ACID IN A TISSUE SAMPLE

Localized detection of RNA in a tissue sample that includes cells is accomplished on an array. The array includes a number of features on a substrate. Each feature includes a different capture probe immobilized such that the capture probe has a free 3′ end. Each feature occupies a distinct position on the array and has an area of less than about 1 mm. Each capture probe is a nucleic acid molecule, which includes a positional domain including a nucleotide sequence unique to a particular feature, and a capture domain including a nucleotide sequence complementary to the RNA to be detected. The capture domain can be at a position 3′ of the positional domain. 1. An array for use in the localized detection of nucleic acid in a tissue sample comprising cells , said array comprising a plurality of features on a substrate , each feature comprising a different capture probe immobilized thereon such that the capture probe has a free 3′ end , each feature occupying a distinct position on the array , each capture probe consisting of a nucleic acid molecule comprising the following domains oriented 5′ to 3′:(i) a positional domain comprising a nucleotide sequence unique to a particular feature; and(ii) a capture domain comprising a nucleotide sequence complementary to the nucleic acid to be detected, wherein the capture domain comprises:(a) a domain that is designed for the selective capture of mRNA; and/or(b) a random or degenerate oligonucleotide sequence; or(c) a sequence specific for a group of genes,wherein the capture probes immobilized on each of the plurality of features on the substrate comprise the same capture domains.2. The array of claim 1 , wherein the capture domain that is designed for the selective capture of mRNA hybridizes to the poly-A tail of mRNA.3. The array of claim 1 , wherein the domain that is designed for the selective capture of mRNA comprises a poly-T DNA oligonucleotide.4. The array of claim 3 , wherein the poly-T DNA oligonucleotide comprises at ...

METHOD AND PRODUCT FOR LOCALIZED OR SPATIAL DETECTION OF NUCLEIC ACID IN A TISSUE SAMPLE

Localized detection of RNA in a tissue sample that includes cells is accomplished on an array. The array include a number of features on a substrate. Each feature includes a different capture probe immobilized such that the capture probe has a free 3′ end. Each feature occupies a distinct position on the array and has an area of less than about 1 mm. Each capture probe is a nucleic acid molecule, which includes a positional domain including a nucleotide sequence unique to a particular feature, and a capture domain including a nucleotide sequence complementary to the RNA to be detected. The capture domain can be at a position 3′ of the positional domain. 1. A method for localized detection of DNA in a tissue sample comprising cells , said method comprising:{'sup': '2', '(a) providing an array comprising a plurality of features on a substrate, each feature comprising a different capture probe immobilized thereon such that the capture probe has a free 3′ end, each feature occupying a distinct position on the array and having an area of less than about 1 mm, each capture probe consisting of a nucleic acid molecule comprising the following domains oriented 5′ to 3′(i) a positional domain comprising a nucleotide sequence unique to a particular feature; and(ii) a capture domain comprising a nucleotide sequence complementary to the DNA to be detected;(b) contacting said array with the tissue sample comprising cells such that the tissue sample contacts a plurality of the features at their distinct positions on the array;(c) hybridizing the DNA present in the tissue sample comprising cells that are complementary to the capture sequences of the capture probes immobilized on the features, such that the DNA is captured by the capture domain of the capture probes in the features;(d) generating DNA molecules from the captured DNA by extending the capture probes enzymatically using: (i) the captured DNA as an extension or ligation template, such that the DNA molecules comprise the ...

METHODS FOR DETERMINING A LOCATION OF A BIOLOGICAL ANALYTE IN A BIOLOGICAL SAMPLE

Provided herein are methods of determining a location of a biological analyte in a biological sample. 1212-. (canceled)213. A method for determining the location of an analyte in a biological sample , the method comprising:(a) providing an array, wherein the array comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises (i) a spatial barcode and (ii) a capture domain;(b) contacting the array with the biological sample, wherein the capture domain of the probe binds to an analyte from the biological sample;(c) contacting a plurality of detectable probes with the analyte bound to the capture probe such that a detectable probe of the plurality of detectable probes interacts with the analyte bound to the capture probe; and(d) determining the location of the detectable probe bound to the analyte, thereby identifying the location of the analyte in the biological sample.214. The method of claim 213 , wherein the capture probe comprises one or more functional domains claim 213 , a cleavage domain claim 213 , a unique molecular identifier claim 213 , and combinations thereof.215. The method of claim 213 , wherein the array comprises one or more features.216. The method of claim 213 , wherein the biological sample is permeabilized under conditions sufficient to allow the analyte to bind the capture probe.217. The method of claim 213 , wherein the detectable probe is a fluorescently labeled detectable probe.218. The method of claim 217 , wherein determining the location of the fluorescently labeled detection probe comprises fluorescence detection.219. The method of claim 218 , wherein a fluorescently labeled probe of the plurality of fluorescently labeled probes comprises nucleic acids.220. The method of claim 219 , wherein the fluorescently labeled probe hybridizes to the analyte.221. The method of claim 220 , wherein the analyte interacts with at least 2 or more fluorescently labeled probes.222. The method of claim 220 , ...

Resolving spatial arrays using deconvolution

Methods for determining a location of a feature on a spatial array include (a) providing an array of features on a substrate, where a feature of the array includes a barcoded oligonucleotide having, in a 5′ to 3′ direction, a spatial barcode, a cleavage domain, and a constant sequence; (b) hybridizing a priming oligonucleotide to the constant sequence; (c) extending the priming oligonucleotide using the barcoded oligonucleotide as a template; and (d) determining all or a portion of a sequence of the extended priming oligonucleotide corresponding to the spatial barcode, or a complement thereof, and a location of the extended priming oligonucleotide, and using the location of the extended priming oligonucleotide to determine the location of the feature on the spatial array.

Methods of Identifying Homologous Genes Using FISH

The present invention relates to methods of hybridizing nucleic acid probes to genomic DNA. 1. A fluorescence in situ hybridization method of distinguishing a first gene in a maternal chromosome from a second gene in a paternal chromosome by single nucleotide polymorphisms which distinguish the first gene from the second gene and wherein the first gene and the second gene are homologs comprisingidentifying a first nucleotide type that is a single nucleotide polymorphism within the first gene,hybridizing a first primer type directly upstream of the first nucleotide type, extending the first primer type across the first nucleotide type in the presence of a first polymerase, first extension nucleotides and a first labeled extension nucleotide complementary to the first nucleotide type, wherein the first labeled extension nucleotide hybridizes to the first nucleotide type,identifying a second nucleotide type that is a single nucleotide polymorphism within the second gene and which is different from the first nucleotide type,hybridizing a second primer type directly upstream of the second nucleotide type, extending the second primer type across the second nucleotide type in the presence of a second polymerase, second extension nucleotides and a second labeled extension nucleotide complementary to the second nucleotide type wherein the second labeled extension nucleotide hybridizes to the second nucleotide type, wherein the first gene is differentially labeled from the second gene.2. The method of further includingidentifying a third nucleotide type that is a single nucleotide polymorphism within the first gene and which is different from the first nucleotide type and the second nucleotide type,hybridizing a third primer type directly upstream of the third nucleotide type, extending the third primer type across the third nucleotide type in the presence of a third polymerase, third extension nucleotides and a third labeled extension nucleotide complementary to the third ...

Devices, systems and methods for ultra-low volume liquid biopsy

Provided herein are devices, systems, kits and methods for obtaining genetic information from cell-free fetal nucleic acids in ultra-low amounts of biological samples. Due to the convenience of obtaining ultra-low amounts of samples, devices, systems, kits and methods can be at least partially employed at a point of need.

Resolving spatial arrays using deconvolution

Methods for determining a location of a feature on an array include: (a) providing a first array with a first plurality of features immobilized on a first substrate; (b) providing a second array with a second plurality of features immobilized on a second substrate; (c) aligning the first array with the second array; (d) hybridizing a first barcoded oligonucleotide of the first array to a second barcoded oligonucleotide of the second array, thereby producing a combined nucleic acid that includes first and second spatial barcodes; (e) determining all or a portion of the sequence of the combined nucleic acid; and (f) identifying the second barcoded oligonucleotide associated with the first barcoded oligonucleotide in the combined nucleic acid, and determining the location of a second feature in the second array.

Nucleic Acid Sample Preparation

This invention relates to the preparation of nucleic acid samples for analysis. The invention may be particularly useful for single stranded samples. Embodiments of the invention involve the attachment of double stranded or hairpin oligonucleotides using template independent polymerase enzymes in the preparation of nucleic acid sequencing libraries. 124.-. (canceled)25. A method comprising: contacting a single stranded oligonucleotide with an enzyme , wherein said enzyme catalyzes an addition of a nucleotide to an end of said single stranded oligonucleotide in a template independent manner , wherein said nucleotide comprises a 5′-triphosphate.26. The method of claim 25 , wherein said enzyme comprises a terminal transferase (TdT).27. The method of claim 26 , wherein said terminal transferase comprises a terminal deoxynucleotidyl transferase (TdT) claim 26 , a polyadenylate polymerase (PAP) or a poly(U)polymerase (PUP).28. The method of claim 25 , wherein said nucleotide comprises a plurality of nucleotides.29. The method of claim 25 , wherein said single stranded oligonucleotide is produced by at least one of (i) a bisulfite treatment; (ii) a chemical or enzymatic cleavage; or (iii) use of an enzyme that is a restriction endonuclease to form said single stranded oligonucleotide.30. The method of claim 25 , wherein a second oligonucleotide strand comprises said nucleotide.31. The method of claim 30 , further comprising associating at least a portion of said single stranded oligonucleotide with at least a portion of said second oligonucleotide strand to form a double-stranded oligonucleotide.32. The method of claim 31 , wherein said second oligonucleotide strand comprises a hairpin.33. The method of claim 32 , wherein said hairpin comprises an extendable 3′-end.34. The method of claim 31 , wherein said second oligonucleotide strand is associated with a solid support.35. The method of claim 34 , wherein said second oligonucleotide strand comprises a moiety for ...

METHODS OF MAKING GENE EXPRESSION LIBRARIES

Provided herein are methods of determining a location of a target nucleic acid in a biological sample. 1. A method of identifying a location of a target nucleic acid in a permeabilized biological sample , the method comprising:(a) generating a cDNA molecule comprising a sequence that is substantially complementary to the target nucleic acid using a reverse transcription primer comprising (i) a sequence that is substantially complementary to a portion of the target nucleic acid and (ii) a first adaptor sequence, wherein the step of generating the cDNA molecule occurs within the permeabilized biological sample;(b) ligating a second adaptor sequence to a 3′ end of the cDNA molecule, wherein the step of ligating is performed within the biological sample;(c) releasing the cDNA molecule from the target nucleic acid, such that the cDNA contacts an array, wherein the array comprises an attached capture probe comprising in a 5′ to a 3′ direction: (i) a spatial barcode and (ii) a capture domain that binds specifically to the second adaptor sequence ligated to the cDNA;(d) extending a 3′ end of the capture probe using the cDNA as a template; and(e) determining (i) all or a part of the sequence of the target nucleic acid, or a complement thereof, and (ii) all or a part of the sequence of the spatial barcode, or a complement thereof, and using the determined sequences of (i) and (ii) to identify the location of the target nucleic acid in the permeabilized biological sample.2. A method of identifying a location of a target nucleic acid in a permeabilized biological sample , the method comprising:(a) generating a cDNA molecule comprising a sequence that is substantially complementary to the target nucleic acid using a reverse transcription primer comprising (i) a sequence that is substantially complementary to a portion of the target nucleic acid and (ii) a first adaptor sequence, wherein the step of generating the cDNA molecule occurs within the permeabilized biological sample;(b ...

SPATIAL MAPPING OF NUCLEIC ACID SEQUENCE INFORMATION

Presented are methods and compositions for spatial detection and analysis of nucleic acids in a tissue sample. The methods can enable the characterization of transcriptomes and/or genomic variations in tissues while preserving spatial information about the tissue. 1. A capture array for spatial detection and analysis of nucleic acids in a tissue sample , comprising a capture site comprising a pair of capture probes immobilized on a surface , wherein a first capture probe of the pair of capture probes comprises a first primer binding region and a spatial address region , and wherein a second capture probe of the pair of capture probes comprises a second primer binding region and a capture region.2. The capture array of claim 1 , wherein the first capture probe does not comprise a gene-specific region.3. The capture array of claim 2 , wherein the second capture probe does not comprise a spatial address region.4. The capture array of claim 1 , wherein the capture site is a plurality of capture sites.5. The capture array of claim 1 , wherein the pair of capture probes in a capture site is a plurality of pairs of capture probes.6. The capture array of claim 5 , wherein each first capture probe in the plurality of pairs of capture probes within the same capture site comprises the same spatial address sequence.7. The capture array of claim 5 , wherein the plurality of pairs of capture probes in different capture sites comprises a different spatial address sequence.8. The capture array of claim 5 , wherein one or more capture sites of the capture array have the same number of first capture probes and of second capture probes.9. The capture array of claim 5 , wherein one or more capture sites of the capture array have more first capture probes than second capture probes.10. The capture array of claim 5 , wherein one or more capture sites of the capture array have more second capture probes than first capture probes.11. The capture array of claim 1 , wherein the capture array ...

In situ combinatorial labeling of cellular molecules

Methods of uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are provided. Kits for uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are also provided. The molecules to be labeled may include, but are not limited to, RNAs and/or cDNAs.

Methods for determining a location of a biological analyte in a biological sample

Provided herein are methods of determining a location of a biological analyte in a biological sample.

Devices, systems and methods for ultra-low volume liquid biopsy

Provided herein are devices, systems, kits and methods for obtaining genetic information from cell-free fetal nucleic acids in ultra-low amounts of biological samples. Due to the convenience of obtaining ultra-low amounts of samples, devices, systems, kits and methods can be at least partially employed at a point of need.

MICROFLUIDIC DEVICE

A microfluidic device comprises a substrate transparent for imaging and having a plurality of spatially defined and separated cell channels having a dimension to accommodate cells in monolayer. A respective first end of the cell channels is in fluid connection with a flow input channel having a first end in fluid connection with a first fluid port and a second end in fluid connection with a second fluid port. A respective second end of the cell channels is in fluid connection with a first end of a respective wash channel having a second end in fluid connection with a flow output channel. The flow output channel is in fluid connection with a third fluid port. The wash channels have a dimension too small to accommodate the cells. 1. A microfluidic device comprising:a substrate having a first set of cell channels and a second set of cell channels;a respective first end of the cell channels of the first set is in fluid connection with a first flow input channel;a respective second end of the cell channels of the first set is in fluid connection with a first flow output channel;a respective first end of the cell channels of the second set is in fluid connection with a second flow input channel;a respective second end of the cell channels of the second set is in fluid connection with a second flow output channel;the cell channels of the first set and of the second set comprise a respective channel restriction in connection with the respective second end to prevent target cells entering the cell channels from reaching the first flow output channel or the second flow output channel.2. The microfluidic device according to claim 1 , whereina first end of the first flow input channel is in fluid connection with a first input port;a second end of the first flow input channel is in fluid connection with a common input port;a first end of the second flow input channel is in fluid connection with a second input port; anda second end of the second flow input channel is in fluid ...

Methods and compositions for nucleic acid analysis

The present invention is directed to methods, compositions and systems for analyzing sequence information while retaining structural and molecular context of that sequence information.

Methods and compositions for nucleic acid analysis

The present invention is directed to methods, compositions and systems for analyzing sequence information while retaining structural and molecular context of that sequence information.

Methods and compositions for nucleic acid analysis

The present invention is directed to methods, compositions and systems for analyzing sequence information while retaining structural and molecular context of that sequence information.

Spatially addressable molecular barcoding

The disclosure provides for methods, compositions, systems, devices, and kits for determining the number of distinct targets in distinct spatial locations within a sample. In some examples, the methods include: stochastically barcoding the plurality of targets in the sample using a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label and a molecular label; estimating the number of each of the plurality of targets using the molecular label; and identifying the spatial location of each of the plurality of targets using the spatial label. The method can be multiplexed.

Spatially addressable molecular barcoding

The disclosure provides for methods, compositions, systems, devices, and kits for determining the number of distinct targets in distinct spatial locations within a sample. In some examples, the methods include: stochastically barcoding the plurality of targets in the sample using a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label and a molecular label; estimating the number of each of the plurality of targets using the molecular label; and identifying the spatial location of each of the plurality of targets using the spatial label. The method can be multiplexed.

Spatially addressable molecular barcoding

The disclosure provides for methods, compositions, systems, devices, and kits for determining the number of distinct targets in distinct spatial locations within a sample. In some examples, the methods include: stochastically barcoding the plurality of targets in the sample using a plurality of stochastic barcodes, wherein each of the plurality of stochastic barcodes comprises a spatial label and a molecular label; estimating the number of each of the plurality of targets using the molecular label; and identifying the spatial location of each of the plurality of targets using the spatial label. The method can be multiplexed.

Detection of nucleic acids

This invention provides compositions, methods, and systems for characterizing, resolving, and quantitating single stranded and double stranded DNA and RNA in-situ. Paired sense and anti-sense probes can signal the presence of double stranded nucleic acids. DNA and RNA can be distinguished in cell and tissue samples by hybridizing with probe sets adapted to highlight differences in these targets in-situ.

Methods and compositions for nucleic acid analysis

본 발명은 서열 정보의 구조적 환경 및 분자 환경을 보유하면서 서열 정보를 분석하기 위한 방법, 조성물 및 시스템에 관한 것이다.

Method and apparatus for volumetric imaging of a three-dimensional nucleic acid containing matrix.

Se proporcionan métodos para formación de imágenes volumétricas de una matriz tridimensional de ácidos nucleicos dentro de una célula; se proporciona un aparato automatizado para secuenciación e imágenes volumétricas de una matriz tridimensional de ácidos nucleicos.

Spatially encoded biological assays

The present invention provides assays and assay systems for use in spatially encoded biological assays. The invention provides an assay system comprising an assay capable of high levels of multiplexing where reagents are provided to a biological sample in defined spatial patterns; instrumentation capable of controlled delivery of reagents according to the spatial patterns; and a decoding scheme providing a readout that is digital in nature.

Method of preparing cell free nucleic acid molecules by in situ amplification

Methods for in situ amplification (ISA) of cfNA, such as cfDNA, in a sample are provided wherein the cfNA in the sample is not subject to a nucleic acid purification step. The methods disclosed may be used to generate an analyzable pool of cfNA present in the sample. The analyzable pool may be used with a variety of analytical techniques to characterize the nucleic acid in the sample. Methods of diagnosis, determining a therapeutic intervention and monitoring of a subject are also provided.

Method of preparing cell free nucleic acid molecules by in situ amplification

method for spatial detection of nucleic acids in a tissue sample

Patent RU2017135434A3

ВУ“? 2017135434” АЗ Дата публикации: 02.10.2019 Форма № 18 ИЗИМ-2011 Федеральная служба по интеллектуальной собственности Федеральное государственное бюджетное учреждение ж 5 «Федеральный институт промышленной собственности» (ФИПС) ОТЧЕТ О ПОИСКЕ 1. . ИДЕНТИФИКАЦИЯ ЗАЯВКИ Регистрационный номер Дата подачи 2017135434/10(061800) 13.04.2016 РСТ/ЕР2016/0580677 13.04.2016 Приоритет установлен по дате: [ ] подачи заявки [ ] поступления дополнительных материалов от к ранее поданной заявке № [ ] приоритета по первоначальной заявке № из которой данная заявка выделена [ ] подачи первоначальной заявки № из которой данная заявка выделена [ ] подачи ранее поданной заявки № [Х] подачи первой(ых) заявки(ок) в государстве-участнике Парижской конвенции (31) Номер первой(ых) заявки(ок) (32) Дата подачи первой(ых) заявки(ок) (33) Код страны 1. 15163481.3 14.04.2015 ЕР Название изобретения (полезной модели): [Х] - как заявлено; [ ] - уточненное (см. Примечания) ПРОСТРАНСТВЕННОЕ КАРТИРОВАНИЕ МОЛЕКУЛЯРНЫХ ПРОФИЛЕЙ ОБРАЗЦОВ БИОЛОГИЧЕСКИХ ТКАНЕЙ Заявитель: КОНИНКЛЕЙКЕ ФИЛИПС Н.В., МГ, 2. ЕДИНСТВО ИЗОБРЕТЕНИЯ [Х] соблюдено [ ] не соблюдено. Пояснения: см. Примечания 3. ФОРМУЛА ИЗОБРЕТЕНИЯ: [Х] приняты во внимание все пункты (см. Примечания) [ ] приняты во внимание следующие пункты: [ ] принята во внимание измененная формула изобретения (см. Примечания) 4. КЛАССИФИКАЦИЯ ОБЪЕКТА ИЗОБРЕТЕНИЯ (ПОЛЕЗНОЙ МОДЕЛИ) (Указываются индексы МПК и индикатор текущей версии) С120 1/68 (2006.01) 5. ОБЛАСТЬ ПОИСКА 5.1 Проверенный минимум документации РСТ (указывается индексами МПК) С120 1/68 5.2 Другая проверенная документация в той мере, в какой она включена в поисковые подборки: 5.3 Электронные базы данных, использованные при поиске (название базы, и если, возможно, поисковые термины): Е-Габгагу, Езрасепев, Рабеагсв, РАТЕМТСОРЕ, КУРТО, МСВТ, ЕМВГ-ЕВ1, Соозе, Соозе эспо[аг, РиБМеа, ОЗРТО, Заепсе) еси 6. ДОКУМЕНТЫ, ОТНОСЯЩИЕСЯ К ПРЕДМЕТУ ПОИСКА Кате- Наименование документа с указанием (где необходимо) частей ...

Methods of generating libraries of nucleic acid sequences for detection via fluorescent in situ sequencing

The present disclosure provides a number of targeted nucleic acid FISSEQ library construction methods. Targeted FISSEQ can exhibit several benefits, such as enhanced sensitivity and/or shorter assay time in the detection, identification, quantification, and/or determining the nucleotide sequence of the target species, relative to “random” or “whole-omic” detection via FISSEQ.

Method of preparing cell free nucleic acid molecules by in situ amplification

Spatially encoded biological assays

The present invention provides assays and assay systems for use in spatially encoded biological assays. The invention provides an assay system comprising an assay capable of high levels of multiplexing where reagents are provided to a biological sample in defined spatial patterns; instrumentation capable of controlled delivery of reagents according to the spatial patterns; and a decoding scheme providing a readout that is digital in nature.

DNA repeat based pre-labelled oligoprobes for rapid and efficient FISH in Panax ginseng

The present invention relates to a nucleic acid probe for detecting the genome of a plant of the genus Ginseng, the nucleic acid probe comprising one or more nucleic acid sequences selected from the group consisting of nucleic acid sequences represented by SEQ ID NOs: 1 to 7; a composition and a kit for detecting the genome of a plant of the genus Ginseng and including the nucleic acid probe; and a method for detecting the genome of ginseng plants. The nucleic acid probe of the present invention or a combination comprising the same has excellent hybridization efficiency and high reproducibility, and enables cost- and time-efficient FISH analysis of major repeat sequences of ginseng plants. Also, because, in a single FISH experiment, one sample slide can be used to simultaneously reveal the chromosomal distribution of 5 different repeat-probes, the present invention can be usefully used to elucidate the genomic structure of plants of the genus ginseng and to analyze the genomes between species.

Systems for analyzing target biological molecules via sample imaging and delivery of probes to substrate wells

The present invention provides assays and assay systems for use in spatially encoded biological assays. The invention provides an assay system comprising an assay capable of high levels of multiplexing where reagents are provided to a biological sample in defined spatial patterns; instrumentation capable of controlled delivery of reagents according to the spatial patterns; and a decoding scheme providing a readout that is digital in nature.

Multiplexed single molecule rna visualization with a two-probe proximity ligation system

SNAIL provides cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids, e.g. at least one, to up to 5, up to 10, up to 15, up to 20 or more transcripts can be simultaneously analyzed, at a rate of up to about 50, 100, 250, 500 or more cells/second. The methods require only two primers for amplification, and may further include a detection primer.

Spatial mapping of nucleic acid sequence information

Presented are methods and compositions for spatial detection and analysis of nucleic acids in a tissue sample. The methods can enable the characterization of transcriptomes and/or genomic variations in tissues while preserving spatial information about the tissue.

Spatial mapping of nucleic acid sequence information

Presented are methods and compositions for spatial detection and analysis of nucleic acids in a tissue sample. The methods can enable the characterization of transcriptomes and/or genomic variations in tissues while preserving spatial information about the tissue.

Devices, systems and methods for ultra-low volume liquid biopsy

Provided herein are devices, systems, kits and methods for obtaining genetic information from cell-free fetal nucleic acids in ultra-low amounts of biological samples. Due to the convenience of obtaining ultra-low amounts of samples, devices, systems, kits and methods can be at least partially employed at a point of need.

Proximity assay with detection based on hybridisation chain reaction (HCR)

The present invention provides a method for detecting an analyte in a sample, said method comprising a) contacting said sample with a set of proximity probes comprising at least first and second proximity probes, which probes each comprise an analyte-binding domain capable of binding directly or indirectly to said analyte and a nucleic acid domain, such that the proximity probes can simultaneously bind, directly or indirectly, to the analyte, wherein i) the nucleic acid domains of said first and second proximity probes comprise regions capable of mediating an interaction involving said domains when under permissive conditions; and ii) the nucleic acid domain of one of said first and second probes comprises an HCR initiator region comprised within a metastable secondary structure such that it is unable to initiate an HCR reaction until released from said metastable secondary structure; b) introducing permissive conditions to allow the nucleic acid domains of said first and second probes to interact with each other when said probes have both bound directly or indirectly to the analyte, wherein said interaction results in unfolding of the metastable secondary structure of the nucleic acid domain of the first or second probe to release a single-stranded HCR initiator region; c) performing an HCR reaction using at least two HCR monomers, wherein the first HCR monomer comprises a region of complementarity to the HCR initiator region and hybridisation of the HCR initiator region to the first HCR polymer begins the HCR reaction to form a polymer; and d) detecting the polymer thereby to detect the analyte.

Spatially distinguished, multiplex nucleic acid analysis of biological specimens

A method for spatially tagging nucleic acids of a biological specimen, including steps of (a) providing a solid support comprising different nucleic acid probes that are randomly located on the solid support, wherein the different nucleic acid probes each includes a barcode sequence that differs from the barcode sequence of other randomly located probes on the solid support; (b) performing a nucleic acid detection reaction on the solid support to locate the barcode sequences on the solid support; (c) contacting a biological specimen with the solid support that has the randomly located probes; (d) hybridizing the randomly located probes to target nucleic acids from portions of the biological specimen; and (e) modifying the randomly located probes that are hybridized to the target nucleic acids, thereby producing modified probes that include the barcode sequences and a target specific modification, thereby spatially tagging the nucleic acids of the biological specimen.

Devices, systems and methods for ultra-low volume liquid biopsy

本文提供用於自超低量生物樣本中之無細胞胎兒核酸獲得遺傳資訊的裝置、系統、套組及方法。由於獲得超低量樣本極為便利，故可以在需要位置處至少部分採用裝置、系統、套組及方法。

Methods of spatially resolved single cell rna sequencing

The present disclosure generally relates to spatial detection of a nucleic acid, such as a genomic DNA or a RNA transcript, in a cell comprised in a tissue sample. The present disclosure provides methods for detecting and/or analyzing nucleic acids, such as chromatin or RNA transcripts, so as to obtain spatial information about the localization, distribution or expression of genes in a tissue sample. The present disclosure thus provides a process for performing "spatial transcriptomics" or "spatial genomics," which enables the user to determine simultaneously the expression pattern, or the location/distribution pattern of the genes expressed or genes or genomic loci present in a single cell while retaining information related to the spatial location of the cell within the tissue architecture.

Methods of using master / copy arrays for spatial detection

This disclosure provides methods for spatial profiling of biological analytes present in a biological sample. Methods include generating feature arrays using a master/copy format using recessed arrays, and methods for using such arrays. For example spatially-tagged analyte capture analytes can be used in spatial detection in methods to determine the location of analytes (e.g., proteins) in biological samples.

DEVICES, SYSTEMS AND METHODS FOR ULTRA-LOW VOLUME LIQUID BIOPSY

Dispositivos, sistemas, kits y métodos para obtener información genética de ácidos nucleicos fetales sin células en cantidades ultra bajas de muestras biológicas. Dada la conveniencia de obtener cantidades ultra bajas de muestras, los dispositivos, sistemas, kits y métodos se pueden emplear, por lo menos parcialmente, donde se los necesita. Devices, systems, kits and methods to obtain genetic information from cell-free fetal nucleic acids in ultra-low quantities from biological samples. Given the convenience of obtaining ultra-low amounts of samples, the devices, systems, kits and methods can be used, at least partially, where they are needed.

Methods of spatially resolved single cell sequencing

The present disclosure generally relates to spatial detection of a nucleic acid, such as a genomic DNA or a RNA transcript, in a cell comprised in a tissue sample. The present disclosure provides methods for detecting and/or analyzing nucleic acids, such as chromatin or RNA transcripts, so as to obtain spatial information about the localization, distribution or expression of genes in a tissue sample. The present disclosure thus provides a process for performing “spatial transcriptomics” or “spatial genomics,” which enables the user to determine simultaneously the expression pattern, or the location/distribution pattern of the genes expressed or genes or genomic loci present in a single cell while retaining information related to the spatial location of the cell within the tissue architecture.

Methods for preparing nucleic acid sequence libraries for detection by fluorescence in situ sequencing

Imaging system hardware

A sample holder includes a first member featuring a first retaining mechanism configured to retain a first substrate that includes a sample, a second member featuring a second retaining mechanism configured to retain a second substrate that includes a reagent medium, and an alignment mechanism connected to at least one of the first and second members, and configured to align the first and second members such that the sample contacts at least a portion of the reagent medium when the first and second members are aligned.

Synthesis of long fish probes

A method comprising: synthesizing a set of overlapping oligonucleotides that comprises probe sequences that hybridize to unique sequences in a chromosome, assembling the overlapping oligonucleotides in a way that produces one or more double stranded polynucleotides that each comprises multiple probe sequences, labeling the one or more double stranded polynucleotides to produce one or more labeled probes, and hybridizing the labeled probes to an intact chromosome, in situ, is provided.

In situ combinatorial labeling of cellular molecules

Methods of uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are provided. Kits for uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are also provided. The molecules to be labeled may include, but are not limited to, RNAs and/or cDNAs.

Methods for performing spatial profiling of biological molecules

Spatially encoded biological assays

The present invention provides assays and assay systems for use in spatially encoded biological assays. The invention provides an assay system comprising an assay capable of high levels of multiplexing where reagents are provided to a biological sample in defined spatial patterns; instrumentation capable of controlled delivery of reagents according to the spatial patterns; and a decoding scheme providing a readout that is digital in nature.

Compositions and methods of rna analysis

The present disclosure relates to compositions and methods of RNA analysis. In particular, the present disclosure provides a method of RNA analysis that includes obtaining a sample, applying one or more multi-partite probes to the sample, where each of the one or more multi-partite probes includes at least two sub-probes, annealing at least one of the applied one or more multi-partite probes to at least one target nucleic acid within the sample, and ligating the at least two sub-probes associated with the at least one annealed multi-partite probe to create a target nucleic acid proxy that can be detected.

Generating spatial arrays with gradients

The present disclosure relates to materials and methods for generating spatial arrays with gradients and using the generated spatial arrays to identify the location of analytes present in a biological sample.

Simultaneous quantification of multiple proteins in user-defined regions of sectioned tissue

본 발명은 특히, 사용자-한정된(user-defined) 조직 영역, 사용자-한정된 세포, 및/또는 세포에서 사용자-한정된 세포내 구조에서 단백질 발현의 동시적인, 다중화된(multiplexed) 검출 및 정량화를 위한 프로브, 조성물, 방법 및 키트에 관한 것이다. The present invention relates, inter alia, to probes for the simultaneous, multiplexed detection and quantification of protein expression in user-defined tissue regions, user-defined cells, and/or user-defined intracellular structures in cells. , compositions, methods and kits.

Methods for preparing cell-free nucleic acid molecules by in situ amplification

Methods of combining the detection of biomolecules into a single assay using fluorescent in situ sequencing

Spatial mapping of molecular profiles of biological tissue samples

FIELD: biotechnology. SUBSTANCE: invention represents a method based on applying patterns of oligonucleotide probes with barcodes on predetermined areas in a region of interest in a tissue sample. Each analyzed nucleic acid can be localized in a specific position within the sample taking into account a barcode. Various printing techniques can be used and various methods for obtaining patterns similar to a regular matrix with a specific pitch can be used, or alternatively object-oriented generation of patterns using certain regions of interest without limitations in form. EFFECT: invention enables spatial mapping of nucleic acids of high-resolution tissue samples without sacrificing the degree of multiplexing available when sequencing the next generation. 13 cl, 3 dwg, 3 tbl, 1 ex

Simultaneous quantification of a plurality of proteins in a user-defined region of a cross-sectioned tissue

The present invention relates to, among other things, probes, compositions, methods, and kits for simultaneous, multiplexed detection and quantification of protein expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.

Antigen-coupled hybridization reagents

The present disclosure provides high-performance hybridization reagents for use in a variety of hybridization assays and other related techniques. The hybridization reagents comprise an oligonucleotide probe and a bridging antigen, wherein the bridging antigen is recognized by a detectable antibody with high affinity. Also provided are compositions comprising panels of hybridization reagents specific for multiple different target nucleic acids and compositions comprising pairs of hybridization reagents and their complementary detectable antibodies. The paired hybridization reagents and detectable antibodies are useful in a variety of hybridization assays, particularly in highly multiplexed assays, where the structure of the bridging antigen is varied in tandem with variation in the detectable antibody, such that a multiplicity of hybridization reagents are provided that are capable of simultaneously detecting a multiplicity of target nucleic acids in a single assay. Also provided are kits comprising the hybridization reagents, methods of hybridization assay using the hybridization reagents of the disclosure, and methods of preparation of the hybridization reagents.

Branched proximity hybridization assay

The invention relates to a method for detecting the proximity of at least two biomolecules using branched DNA technology. The assay is called branched proximity hybridization assay.

Devices, systems and methods for ultra-low volume liquid biopsy

Provided herein are devices, systems, kits and methods for obtaining genetic information from cell-free fetal nucleic acids in ultra-low amounts of biological samples. Due to the convenience of obtaining ultra-low amounts of samples, devices, systems, kits and methods can be at least partially employed at a point of need.

Spatial mapping of nucleic acid sequence information

Presented are methods and compositions for spatial detection and analysis of nucleic acids in a tissue sample. The methods can enable the characterization of transcriptomes and/or genomic variations in tissues while preserving spatial information about the tissue.

Matrix imprinting and clearing

The present invention generally relates to systems and methods for imaging or determining nucleic acids or other desired targets, for instance, within cells or tissues. In one aspect, a sample is exposed to a plurality of nucleic acid probes that are determined within the sample. In some cases, however, background fluorescence or off-target binding may make it more difficult to determine properly bound nucleic acid probes. Accordingly, other components of the samples that may be contributing to the background, such as proteins, lipids, and/or other non-targets, may be "cleared" from the sample to improve determination. However, in certain embodiments, nucleic acids or other desired targets may be prevented from also being cleared, e.g., using polymers or gels within the sample. Other aspects are generally directed to compositions or kits involving such systems, methods of using such systems, or the like.

Multiplex single-molecule RNA visualization using a dual-probe proximity ligation system

SNAIL提供了单细胞中的特定核酸的有成本效益的检测，并且可与流式细胞术组合以针对多个核酸同时分析大量细胞，例如，能够以高达约50、100、250、500或更多个细胞/秒的速率同时分析至少一种至高达5种、高达10种、高达15种、高达20种或更多种转录物。所述方法仅需要两种引物用于扩增，且可进一步包括检测引物。

Method for detecting DNA end (s) and use thereof

본 발명은 하기 단계 I 내지 III, 및 단계 I 내지 III 각각의 하위-단계 a 내지 h 중 적어도 하나를 포함하는, 생물학적 물질에서 DNA 말단(들)을 검출하는 방법에 관한 것으로서, I. 물질의 제조: a. 생물학적 물질을 고정 및/또는 투과 및/또는 용해 및/또는 단리 및/또는 분획화 및/또는 부동화시키는 단계, b. DNA 말단(들)의 접근성을 증가시키는 단계, c. 상기 생물학적 물질에서 분자 유형 2 내지 6에 대한 비특이적인 결합 부위(들)를 차단하는 단계를 포함함; II. DNA 말단(들)의 가공: d. 화학적 또는 물리적 가공에 의한 DNA 말단(들)의 변형, 뒤이어 분자 유형 1을 촉매적 또는 비촉매적 수단에 의해 DNA 말단(들)에 결합시키는 단계; 생물학적 물질에서 분자 유형 2 내지 6에 대한 비특이적인 결합 부위(들)를 차단하는 단계를 포함함; III. 변형된 DNA 말단(들)의 인지 및 검출: 단계 II 유래의 생물학적 물질을, 롤링 서클 증폭(RCA; rolling circle amplification) 반응을 유발하는 단계를 가능하게 하는 방식으로 분자 유형 1에 결합하는 적어도 2개의 분자 유형 2 및 3과 함께 인큐베이션하는 단계, g. DNA 말단(들)을: i. 선택적으로 적합한 분자 유형 4 및/또는 5를 분자 유형 2 및 3과 접촉시키는 단계로서, 상기 분자 유형 4 및/또는 5는 올리고뉴클레오타이드 유형 1과 공액되는 단계, ii. 올리고뉴클레오타이드 유형 2 및 효소 리가제(ligase)를 첨가하여, 상기 첨가된 올리고뉴클레오타이드 유형 2를 분자 유형 4 및/또는 5에 이미 연결된 올리고뉴클레오타이드 유형 1에, 또는 분자 유형 2 및 3이 올리고뉴클레오타이드 유형 1에 연결된다면 상기 분자 유형 2 및 3에 혼성화시키고, 후속적으로 올리고뉴클레오타이드 유형 2의 DNA 결찰을 수행하는 단계, iii. 효소 폴리머라제 및 뉴클레오타이드 용액을 첨가하여 롤링 서클 증폭(RCA) 반응을 가능하게 함으로써 증폭을 수행하고, 분자 유형 6을 첨가하여 상기 분자 유형 6과 RCA 반응의 수득된 생성물의 후속적인 혼성화를 가능하게 하는 단계에 의해 검출하는 단계, h. 분자 유형 6을 검출하는 단계; 이러한 단계 I의 1개 초과의 하위-단계 a 내지 c가 수행될 때, 이들 하위-단계는 임의의 순서로 발생할 수 있다. 본 발명은 또한, 생물학적 물질에서 단일 DNA 말단(들)의 존재 및 위치를 표시하기 위한 롤링 서클 복제의 용도, 및 DNA 말단(들)의 검출을 위한 상기 언급된 방법의 용도에 관한 것이다.

Method and Apparatus for Volumetric Imaging

Methods of volumetric imaging of a three-dimensional matrix of nucleic acids within a cell is provided. An automated apparatus for sequencing and volumetric imaging of a three-dimensional matrix of nucleic acids is provided.

Methods and kits

Simultaneous quantification of a plurality of proteins in a user-defined region of a cross-sectioned tissue

Composiciones y métodos de análisis de ARN

La presente divulgación se refiere a composiciones y métodos de análisis de ARN. En particular, la presente divulgación proporciona un método de análisis de ARN que incluye obtener una muestra, aplicar una o más sondas multipartitas a la muestra, donde cada una de las una o más sondas multipartitas incluye al menos dos subsondas, hibridación al menos una de las una o más sondas multipartitas aplicadas a al menos un ácido nucleico diana dentro de la muestra, y ligar las al menos dos subsondas asociadas con al menos una sonda multipartita hibridada para crear un ácido nucleico diana proxy que se puede detectar. (Traducción automática con Google Translate, sin valor legal)

Analysis of target molecules within a sample via hybridization chain reaction

Analysis of target molecules within a sample via hybridization chain reaction

In situ combinatorial labeling of cellular molecules

Methods of uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are provided. Kits for uniquely labeling or barcoding molecules within a nucleus, a plurality of nuclei, a cell, a plurality of cells, and/or a tissue are also provided. The molecules to be labeled may include, but are not limited to, RNAs and/or cDNAs.

Rna分析的组合物和方法

本公开涉及RNA分析的组合物和方法。特别地，本公开提供一种RNA分析方法，该方法包括获得样品；将一种或多种多歧探针施加至该样品，其中该一个或多个多歧探针包括至少两个子探针；将所施加的一个或多个多歧探针的至少一个退火为该样品内的至少一个靶标核酸；以及将结合该至少一个退火的多歧探针的至少两个子探针连接，以创造可被检测的靶标核酸替代物。

Method and kits for multiplexed fluorescent microscopy

Provided is a method for multiplexed fluorescence microscopy comprising contacting a fixed sample with a set of binding agent-T-oligonucleotide conjugates to allow the binding agents to bind to any binding partners present in the sample, wherein the set comprises a plurality of binding agents having different specificities and the sequence of the T-oligonucleotide is unique to the binding agent to which it is conjugated, contacting the sample and any bound binding agents resulting from step a with a FRET- oligonucleotide, illuminating the sample with a wavelength to cause excitation of the FRET- oligonucleotide's emitter molecule, and observing the fluorescent kinetic profile of the sample at the FRET-oligonucleotide emitter molecule's emission wavelength at one or more pixels over time, wherein the FRET-oligonucleotide can hybridise to multiple T-oligonucleotides in the set, to form multiple pairs, and wherein the dissociation and reassociation between each different pair generates a fluorescent kinetic profile that is unique within that set to that pair. Also provided are associated kits, sets of binding agent T-oligonucleotide conjugates and corresponding FRET-oligonucleotides, and methods of designing such sets.

Imaging system hardware

A sample holder includes a first member featuring a first retaining mechanism configured to retain a first substrate that includes a sample, a second member featuring a second retaining mechanism configured to retain a second substrate that includes a reagent medium, and an alignment mechanism connected to at least one of the first and second members, and configured to align the first and second members such that the sample contacts at least a portion of the reagent medium when the first and second members are aligned.

与指数辐亮度拴系的连锁放大

本文公开了用于与指数辐亮度拴系的连锁放大的组合物用于信号放大。本文还公开了用于与指数辐亮度拴系的连锁放大的试剂盒用于信号放大。本文还公开了用于与指数辐亮度拴系的连锁放大的方法用于信号放大。

Spatial Mapping Of Nucleic Acid Sequence Information

Presented are methods, compositions and bead arrays for spatial detection and analysis of nucleic acids in a tissue sample. The methods can enable the characterization of transcriptomes and/or genomic variations in tissues while preserving spatial information about the tissue.