406 results about "Ligation" patented technology

The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template. The cycles comprise steps of extension, ligation, and, preferably, cleavage. In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages. In certain embodiments the methods make use of extension probes containing an abasic residue or a damaged base and employ agents appropriate to cleave linkages between a nucleoside and an abasic residue and/or agents appropriate to remove a damaged base from a nucleic acid. The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families. In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle. In certain embodiments the sequencing reactions are performed on templates attached to beads, which are immobilized in or on a semi-solid support. The invention further provides sets of labeled extension probes containing phosphorothiolate linkages or trigger residues that are suitable for use in the method. In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides. The invention further provides efficient methods for preparing templates, particularly for performing sequencing multiple different templates in parallel. The invention also provides methods for performing ligation and cleavage. The invention also provides new libraries of nucleic acid fragments containing paired tags, and methods of preparing microparticles having multiple different templates (e.g., containing paired tags) attached thereto and of sequencing the templates individually. The invention also provides automated sequencing systems, flow cells, image processing methods, and computer-readable media that store computer-executable instructions (e.g., to perform the image-processing methods) and/or sequence information. In certain embodiments the sequence information is stored in a database.

Methods and compositions are provided for circularizing target sequences in a sample. In particular, ligation oligonucleotides are employed to selectively hybridize with the target such that the target can be ligated into a closed circular target. Rolling circle amplification can then be performed directly on the target sequence for subsequent detection and analysis.

The present invention teaches a novel approach to detecting and/or analyzing nucleic acid sequences. Generally, the invention relates to detecting and/or analyzing nucleic acid sequences using microsphere-based assays. More specifically, the invention relates to detecting and/or analyzing nucleic acid sequences using microsphere-based oligonucleotide ligation multiplexed assays. The present invention also provides methods and kits for performing microsphere-based oligonucleotide ligation assays, which include bound probes attached to addressable microspheres and free probes bearing a detectable label.

A method for amplifying a plurality of target sequences that minimizes amplification artefacts is provided. A sample of interest is fragmented into fragments, where each fragment that includes a target sequence has at least one defined end sequence. Selector constructs, all comprising a primer pair motif and each individual selector comprising one or two protruding ends complementary to the defined end sequences of the fragments containing the target sequences, are brought in contact with the fragments. After ligation, the selected target sequences are amplified in parallel using a primer-pair specific for the primer-pair motif common to the selectors.

A pair of oligonucleotide probes comprising a first oligonucleotide probe that comprises a first clamp section that is capable of hybridising to a second clamp section of a second oligonucleotide probe and a first target section that is capable of hybridising to a first section of a target DNA sequence to be detected, a second oligonucleotide probe that comprises a second clamp section that is capable of hybridising to the first clamp section of the first oligonucleotide probe and a second target section that is capable of hybridising to a second section of the target DNA sequence to be detected.

Exemplary methods, procedures, computer-accessible medium, and systems for base-calling, aligning and polymorphism detection and analysis using raw output from a sequencing platform can be provided. A set of raw outputs can be used to detect polymorphisms in an individual by obtaining a plurality of sequence read data from one or more technologies (e.g., using sequencing-by-synthesis, sequencing-by-ligation, sequencing-by-hybridization, Sanger sequencing, etc.). For example, provided herein are exemplary methods, procedures, computer-accessible medium and systems, which can include and/or be configured for obtaining raw output from a sequencing platform configured to be used for reading fragment(s) of genomes, obtaining reference sequences for the genomes obtained independently from the raw output, and generating a base-call interpretation and/or alignment using the raw output and the reference sequences. For example, a score function can be determined based on information associated with the sequencing platform that can be used to analyze polymorphisms based on the base-call interpretation and/or alignment.

The invention provides compositions and methods for ligating single stranded nucleic acids wherein the ligation is based on fast, efficient, and low-sequence bias hybridization of an acceptor molecule with a donor molecule. In one embodiment, the structure of the donor molecule comprises a stem-loop intramolecular nucleotide base pairing (i.e., hairpin) and a 3′-overhang region such that the overhang is able to hybridize to nucleotides present in the 3′ end of the acceptor molecule.

Currently, the circularization of small RNAs is broadly regarded as an obstacle in ligation-related assays and explicitly avoided while short lengths of linear RNA targets is broadly recognized as a factor limiting use of conventional primers in PCR-related assays. In contrast, the disclosed invention capitalizes on circularization of small RNA targets or their conjugates with oligonucleotide adapters. The circular RNA templates provide amplification of the target sequences via synthesis of multimer nucleic acids that can be either labeled for direct detection or subjected to PCR amplification and detection. Structure of small circular RNAs and corresponding multimeric nucleic acids provide certain advantages over current methods including flexibility in design of conventional RT and PCR primers as well as use of 5′-overlapping dimer-primers for efficient and sequence-specific amplification of short target sequences. Our invention also reduces number of steps and reagents while increasing sensitivity and accuracy of detection of small RNAs with both 2′OH and 2′-OMe at their 3′ ends. Our invention increase sensitivity and specificity of detection of microRNAs and other small RNAs with both 2′OH and 2′-OMe at their 3′ ends while allowing us to distinguish these two forms from each other.

Aspects of the invention relate to methods and kits for assessing cancer. Some aspects of the invention relate to methods and kits for preparing a sample library for sequencing. Some aspects of the invention relate to methods and kits for allele detection. Some aspects of the invention relate to high efficiency ligation methods and kits. Some aspects of the invention relate to sensitive detection of amplicons.

Disclosed are compositions and a method for of amplifying nucleic acid sequences useful for detecting the presence of molecules of interest. The method is useful for detecting specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of a DNA ligation operation, an amplification operation, and a detection operation. The DNA ligation operation circularizes a specially designed nucleic acid probe molecule. This operation is dependent on hybridization of the probe to a target sequence and forms circular probe molecules in proportion to the amount of target sequence present in a sample. The amplification operation is rolling circle replication of the circularized probe. A single round of amplification using rolling circle replication results in a large amplification of the circularized probe sequences. Following rolling circle replication, the amplified probe sequences are detected and quantified using any of the conventional detection systems for nucleic acids such as detection of fluorescent labels, enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels. Because, the amplified product is directly proportional to the amount of target sequence present in a sample, quantitative measurements reliably represent the amount of a target sequence in a sample. Major advantages of this method are that the ligation step can be manipulated to obtain allelic discrimination, the DNA replication step is isothermal, and signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme. In multiplex assays, the primer oligonucleotide used for the DNA polymerase reaction can be the same for all probes. Also described are modes of the method in which additional amplification is obtained using a cascade of strand displacement reactions.

The present invention provides a method for determining the sequence of a nucleic acid by successive cycles of duplex extension along a single-stranded template. This cycle includes the steps of: extension, ligation, preferably cleavage. In certain embodiments, the method utilizes an extension probe containing a phosphorothioate linkage and utilizes a substance suitable for cleaving such linkage. In certain embodiments, the method utilizes extension probes containing abasic residues or damaged bases, and utilizes substances suitable for cleaving linkages between nucleosides and abasic residues and/or suitable for Removes substances that damage bases in nucleic acids. The present invention provides methods for determining sequence information using at least two differentially labeled probe families. In certain embodiments, the method obtains less than 2 bits of information per cycle from each of the plurality of nucleotides of the template. In certain embodiments, the sequencing reaction is performed on templates attached to beads immobilized in or on a semi-solid support. The invention also provides labeled extension probe sets containing phosphorothioate linking or priming residues suitable for use in this method. In addition, the present invention includes removing the starting oligonucleotide and the extending strand and performing subsequent reactions with different starting oligonucleotides, thereby performing multiple sequencing reactions on one template. The invention also provides an effective method for preparing templates, especially for parallel sequencing of multiple different templates. The invention also provides methods for performing ligation and cleavage. The invention also provides novel libraries of nucleic acid fragments containing paired tags, methods for preparing microparticles to which multiple different templates (eg, containing paired tags) are attached, and for individually sequencing these templates. The invention also provides automated sequencing systems, flow chambers, image processing methods, and computer-readable media storing computer-executable instructions (such as performing image processing methods) and/or sequence information. In certain embodiments, sequence information is stored in a database.

The invention provides a method or a kit for capturing and labeling one or more target sequences of multiple samples based on high-throughput sequencing. A novel labeling technical scheme is provided by integrating a multiplex amplification technology, a ligation technology and a PCR-index (polymerase chain reaction-index) technology; the target sequences are amplified by virtue of specific capturing primers; by virtue of a ligation reaction, an adaptor, which is provided with a U base and has a known sequence, is introduced to each of two ends of a PCR product; the U bases are cut open by virtue of a USER enzyme; PCR primers are designed in accordance with the known sequences of the adaptors; tag primers are separately introduced to two ends of the PCR product by virtue of a PCR reaction; sample information of the PCR product can be specifically obtained by virtue of the sequences of the tag primers which are introduced to two ends of the PCR product; and then, in accordance with the sequence of the capturing primer of each of the target sequences, a corresponding sequencing result is correspondingly applied to each of the target sequences of the samples.