Novel assay for nucleic acid analysis
AssigneeSyngenta Participations AG
InventorsLi Yang, Xun Wang, Tong Zhu, Liang Shi
Life of this patent
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Abstract
Currently, three technologies are utilized for analysis of gene expression: hybridzation-based technologies, PCR-based technologies, and sequence-based technologies The present invention provides a method for analyzing the presence and/or amount of a specific nucleic acid using a solid support and a capture probe complementary to a region of a target nucleic acid, and polymerizing a labeled extension complementary to the target nucleic acid The invention provides a method of analysis of all types of nucleic acids, and can be used to study multiple genes in a single assay using different capture probes conjugated to different class of microspheres that can be mixed in any desired combination.
Claims66
A method of analysis of a nucleic acid sample, comprising the steps of:
- (a) providing a substrate comprising a solid support and a capture probe linked thereto, the capture probe having a sequence complementary to a first segment of a sequence of a single-stranded target nucleic acid;
- (b) contacting the substrate with a nucleic acid sample, under conditions suitable for hybridization between the capture probe and the target nucleic acid, wherein upon the hybridization at least a second segment of the sequence of the target nucleic acid remains single stranded;
- (c) exposing the substrate to conditions suitable for complementing at least a second segment of the target nucleic acid, wherein the complementing nucleic acid comprises nucleotides having a label capable of enhancing sensitivity of detection of the complementing nucleic acid;
- and
- analyzing the label to determine presence or absence of the target nucleic acid in the nucleic acid sample.
A method of screening for changes in the expression or regulation of a target nucleic acid in a biological system, comprising the steps of:
- (a) treating the biological system with a substance;
- or subjecting the biological system to changed environmental conditions;
- (b) extracting a nucleic acid sample from the biological system;
- (c) providing a substrate comprising a solid support and a capture probe linked thereto, the capture probe having a sequence complementary to a first segment of a sequence of a single-stranded target nucleic acid;
- (d) contacting the substrate with the nucleic acid sample extracted from the biological system, under conditions suitable for hybridization between the capture probe and the target nucleic acid, wherein upon the hybridization a second segment of the sequence of the target nucleic acid remains single stranded;
- (e) exposing the substrate to conditions suitable for complementing at least a second segment of the target nucleic acid, wherein the complementing nucleic acid comprises nucleotides having a label capable of enhancing sensitivity of detection of the complementing nucleic acid and wherein the complementation is preferably achieved by polymerizing an extension complementary to the second segment of the target nucleic acid, wherein the extension comprises nucleotides having a label capable of enhancing sensitivity of detection of the extension;
- (f) analyzing the label to determine presence or absence of the target nucleic acid in the nucleic acid sample;
- and
- (g) determining changes in the expression or regulation of the target nucleic acid in the biological system.
A system of gene expression analysis, the system comprising a microbead having at least two different fluorochromes, the system further comprising at least one capture probe linked to the microbead, the capture probe having a sequence complementary to a first segment of a sequence of a target nucleic acid, the system also comprising a labeled probe complementary to at least a second segment of the sequence of the target nucleic acid, wherein the labeled probe comprises a label capable of enhancing sensitivity of detection thereof.
A diagnostic kit suitable for diagnosis of a particular physiological state of an organism, comprising a solid support and a capture probe linked to the solid support, wherein the capture probe is complementary to a first segment of a target nucleic acid associated with the physiological state.
A method for marker assisted breeding comprising the steps of:
- providing a substrate comprising a solid support and a capture probe linked thereto, the capture probe having a sequence complementary to a first segment of a sequence of a target nucleic acid, wherein the target nucleic acid is correlated with a trait of interest in a breeding program;
- contacting the substrate with a nucleic acid sample from an individual or population in the breeding program, under conditions suitable for hybridization between the capture probe and the target nucleic acid;
- probing a second segment of the target nucleic acid to detect presence or absence of the target nucleic acid;
- and
- determining desirability of the individual or population for the breeding program, based on the presence or absence of the target nucleic acid, whereby the individual is used for marker assisted breeding.
A method of determining effectiveness of a capture probe, comprising the steps of:
- providing a substrate comprising a solid support and a capture probe linked thereto, the capture probe having a sequence complementary to a first segment of a sequence of a single-stranded target nucleic acid;
- contacting the substrate with a nucleic acid sample, under conditions suitable for hybridization between the capture probe and the target nucleic acid, wherein upon the hybridization at least a second segment of the sequence of the target nucleic acid remains single stranded;
- exposing the substrate to conditions suitable for polymerizing an extension complementary to the second segment of the target nucleic acid, wherein the extension comprises nucleotides having a label capable of enhancing sensitivity of detection of the extension;
- and
- analyzing the label quantitatively to determine effectiveness of the capture probe in capturing the target nucleic acid.
A method of analysis of a nucleic acid sample, comprising the steps of providing a substrate comprising a solid support and a capture probe linked thereto;
- providing a single-stranded target nucleic acid sample, comprising at least a first segment, a second segment, and a third segment, wherein the capture probe has a sequence complementary to a portion of one of the segments;
- contacting the substrate with the nucleic acid sample, under conditions suitable for hybridization between the capture probe and the target nucleic acid, wherein upon the hybridization at least two of the segments of the nucleic acid sample remain single stranded,
- contacting the substrate with at least one labeled probe, under conditions suitable for hybridization between the labeled probe and a portion of a single stranded segment of the nucleic acid sample, wherein the labeled probe comprises a nucleic acid sequence complementary to at least a portion of the single stranded segment of the nucleic acid sample;
- and
- analyzing the label to determine presence or absence of the target nucleic acid in the nucleic acid sample.
Dependencies are inferred from the claim text.