43 results about "Primer extension" patented technology

To provide a gene testing method and a gene testing kit for analyzing a variable region of a TCR or a BCR in single-cell analysis.SOLUTION: A method including a step of capturing mRNA eluted from a single cell using an RT probe immobilized on a solid-phase surface and including a common sequence, a cell identification barcode, a first molecular identification barcode, and an oligo(dT) sequence, a step of performing a reverse transcription reaction in a reverse transcription reaction solution containing a TSO using the mRNA as a template to generate cDNA having a sequence added to a 3' end, a step of degrading the mRNA, a step in which a primer for inserting a second molecular identification barcode binds to a constant region adjacent to a variable region of a TCR or a BCR and includes the second molecular identification barcode, a step of ligating cDNA extended from the primer with a ligase, a step of amplifying a fragment including the second molecular identification barcode and the variable region of the TCR or the BCR using a primer, a step of performing sequence analysis, and a step of associating sequences.SELECTED DRAWING: Figure 2

The present invention provides a method for simultaneously amplifying multiple target nucleic acid regions in a single reaction volume, and a method for selecting a primer library to be used in such amplification. Furthermore, the present invention provides a primer library having desired properties, such as minimal formation of amplification primer dimers or other non-target amplification products. [Solution] For example, a method for amplifying a target gene locus in a nucleic acid sample is provided, comprising the steps of (a) contacting the nucleic acid sample with a test primer library that simultaneously hybridizes to at least 1,000 different target gene loci to generate a reaction mixture, and (b) subjecting the reaction mixture to primer extension reaction conditions to generate an amplification product containing a target amplification product.

Provided is a conditional multiplex QPCR method. A QPCR amplification system, for each DNA sample, comprises a set of conditional primers 1 and 2. Forward conditional primers 1 and 2 are paired with a template strand, and reverse conditional primers 1 and 2 are paired with a complementary strand of the template strand. A conditional primer 2 is located downstream of a conditional primer 1, the 5' end of the conditional primer 2 is connected to an amplification primer of a DNA fragment to be detected, and the amplification primer is not paired with an upstream sequence of a binding site between the conditional primer 2 and the template strand. In the method, only when both the conditional primers 1 and 2 are bound to the template strand and the conditional primer 1 extends to the 5' end of the conditional primer 2, the DNA polymerase cleaves the amplification primer of the DNA fragment to be detected, which is free at the 5' end of the conditional primer 2, into an amplification system. The method reduces the possibility of unnecessary pairing between the amplification primer of the DNA fragment to be detected and other different DNA samples, thereby improving the specificity of the amplification reaction.

The disclosure relates to a method for sequencing of a DNA strand, the method includes (S200) providing a double-stranded DNA fragment with a first strand and a second strand, the DNA strand to be sequenced corresponding to the first strand or the second strand, (S300A) connecting the first and second strands with a first hairpin oligonucleotide and a second hairpin oligonucleotide, (S500A) amplifying the ligated product obtained in step (S300A) using rolling circle amplification (RCA), (S600A) amplifying the ligated product obtained in step (S300A) using RCA or the amplicons obtained in step (S500A) using primer extension using a first group of redox-modified nucleotides and a second group of redox-modified nucleotides, and (S800A) determining the sequence of the DNA strand by sequencing the amplicons (80) obtained in step (S600A) using an electrochemical sequencing method.

Disclosed herein include systems, methods, compositions, and kits for labeling nucleic acid targets in a sample. In some embodiments, nucleic acid targets (e.g., mRNAs) are initially barcoded on the 3′ end and are subsequently barcoded on the 5′ end following a template switching reaction and intermolecular and / or intramolecular hybridization and extension. 5′- and / or 3′-barcoded nucleic acid targets can serve as templates for amplification reactions and / or random priming and extension reactions to generate a sequencing library. The method can comprise generating a full-length sequence of the nucleic acid target by aligning a plurality of sequencing reads. Immune repertoire profiling methods are also provided in some embodiments.

Disclosed are DNA polymerases having increased 5′-3′ strand displacement activity and substantially reduced 5′-3′ exonuclease and endonuclease activity relative to a corresponding, unmodified polymerase. The polymerases are useful in a variety of disclosed primer extension methods. Also disclosed are related compositions, including recombinant nucleic acids, vectors, and host cells, which are useful, e.g., for production of the DNA polymerases.

Described herein are methods synchronizing sequencing primers within a sequencing cluster and methods of generating long-range sequencing reads. The methods can include hybridizing primers to polynucleotide copies within a sequencing cluster; extending the primers through a first region of the polynucleotide copies using labeled nucleotides according to a sequencing flow order; extending the primers through a second region of the polynucleotide copies using one or more re-phasing flow steps that each include at least two different types of nucleotide bases; and extending the primers through a third region of the polynucleotide copies using labeled nucleotides according to the sequencing cycle. The rephasing flow steps may be initiated after a predetermined number of sequencing flow steps, after a measured sequencing signal strength falls below a predetermined sequencing signal strength threshold, or a measured sequencing signal-to-noise ratio falls below a sequencing signal-to-noise ratio threshold.

Improved methods and compositions are provided herein for primer extension target enrichment of target polynucleotides.

This invention discloses a primer-probe combination, method, and application for detecting pathogenic microorganisms. The nucleic acid sequences of the primers include those shown in SEQ ID NO.1-SEQ ID NO.42; the nucleic acid sequences of the fluorescent probes include those shown in SEQ ID NO.43-SEQ ID NO.63. This invention, through a primer design approach combining conserved and specific primers, and based on one-step quantitative real-time PCR technology, achieves rapid differentiation of pathogen types in patients. It has a wide target coverage, high detection efficiency, and ensures stable binding of the probe to the target fragment during primer extension, resulting in high accuracy and effectively avoiding cross-contamination and aerosol contamination.

Methods and compositions for multi-step primer extension reactions, such as multiplex polymerase chain reaction (PCR) and reverse transcriptase PCR, are described. [Solution] The primer extension steps are performed in a closed container without opening the container between steps. The multi-step primer extension method and composition utilizes an earlier stage primer in an earlier stage and a later stage primer in a later stage, where the later stage primer is blocked from extension during the earlier stage. The blocked primers of this technology contain photocleavable protecting groups that are substantially inactive until the protecting group is cleaved by exposure to ultraviolet light. The blocked primers can be activated by ultraviolet light without opening the container.

To provide a method for enrichment of at least one target nucleic acid in a library of nucleic acids.SOLUTION: A first oligonucleotide is hybridized to a target nucleic acid in library of nucleic acids having first and second adapters. The hybridized first oligonucleotide is extended with a first polymerase, thereby producing a first primer extension complex including the target nucleic acid and the extended first oligonucleotide. The first primer extension complex is captured, enriched relative to the library of nucleic acids, and a second oligonucleotide is hybridized to the target nucleic acid. The hybridized second oligonucleotide is extended with a second polymerase, thereby producing a second primer extension complex including the target nucleic acid and the extended second oligonucleotide, and further liberating the extended first oligonucleotide from the first primer extension complex.SELECTED DRAWING: Figure 1

Presented arc two new methods, single primer extension reaction (SSPER) and reduce recycle PCR (rrPCR). These methods have a step that easily removes the oligonucleotide primer(s) after the first reaction, thus, allowing for the addition of a second reaction in chronological sequence to generate and isolate the appropriate DNA product with the site-directed mutation(s). A high efficiency of the methods is demonstrated by generating all ten site-directed mutations and six paired combinations of these mutations on four plasmid DNA templates ranging from 10 to 12 kb and 57 to 59% GC-content at a rate of 50-100%. The methods are i) highly accurate allowing for screening of plasmids by DNA sequencing, ii) streamlined to generate the mutations within a single day, iii) cost-effective in requiring only two primers and two enzymes (Dpnl and a proofreading DNA polymerase), iv) straightforward in primer design, and v) applicable for both large and small plasmids.

The present disclosure is directed to compositions, kits, and methods of target enrichment by unidirectional primer extension, whereby the compositions, kits, and methods utilize both poison primers and target capture primers.

The present invention discloses a method for amplifying double-stranded circular nucleic acids, comprising: mixing and incubating a CRISPR RNP complex with a double-stranded circular nucleic acid, wherein the double-stranded circular nucleic acid includes a first strand and a second strand, wherein the sgRNA can specifically bind to a target site on the first strand, and wherein the Cas9 nickase can cleave the second strand; adding a nuclease exonuclease to digest the second strand to obtain a nicked strand; separating the CRISPR RNP complex from the first strand; and using the nicked strand as a primer and the first strand as a template, using a polymerase to extend a nucleic acid complementary to the template from the primer. The double-stranded circular nucleic acid amplification method of the present invention has strong specificity and simple operation, can obtain a long single-stranded rolling circle amplification product, and has broad application prospects.

Provided herein are products and processes for detecting the presence or absence of minor nucleic acid species in a sample containing a mixture of minor nucleic acid species and one or more major nucleic acid species, where the amount (frequency or copy number) of the minor nucleic acid species is less than that of the major nucleic acid species. Certain methods include amplifying the mixture and extending the resulting amplicons using chain terminating reagents and extension primers that specifically hybridize to the amplicons, where a chain terminating reagent specific for the major nucleic acid species has a concentration that is less than a chain terminating reagent that is specific for a minor nucleic acid species. Skewing the concentrations of the chain terminating reagents in favor of high concentrations of the chain terminating reagents specific for the minor nucleic acid species relative to a chain terminating reagent specific for a major nucleic acid species improves the detection limit (sensitivity) of detecting minor nucleic acid species present at low frequency or copy number in the mixture. In addition, the signals generated from the extension product of the major nucleic acid species amplicon can serve as a positive control and permit quantification of the minor nucleic acid species relative to the major nucleic acid species in the mixture.

The present disclosure relates to compounds, methods, and compositions for improving nucleic acid polymerization, including DNA replication by in vitro primer extension to produce polymers for, e.g., nanopore-based single molecule sequencing of DNA templates. Provided are nucleic acid polymerase reaction compositions with polymerization-enhancing moieties that allow for enhanced DNA polymerase activity on nucleotide analogs, thereby improving the length of primer extension products for sequencing applications.

Methods for preparing a solid surface for oligonucleotide sequencing including modifying a sequence of an oligonucleotide to be sequenced on a solid surface, such as on a flow of a sequencer. The modification may disrupt a sequence in a region known to produce a sequence specific error or may provide a bioinformatic tag upstream of a region of interest. The modification may be performed by hybridizing a template oligonucleotide to a surface primer, hybridizing a tagging oligonucleotide to a target region of the target oligonucleotide, where the tagging oligonucleotide includes a sequence different from the sequence of the target region, and extending the surface primer from a 3 ' end primer using a target oligonucleotide as a template, and extending the tagging oligonucleotide from a 3 ' end using the target oligonucleotide to produce the copy oligonucleotide having a sequence complementary to the target oligonucleotide except for in the tag region.

The present disclosure provides a method of enriching a variant nucleic acid molecule from a population of nucleic acid molecules, said method comprising contacting a population of nucleic acid molecules with a primer in a reaction mixture, incubation of the reaction mixture for hybridization of the primer with a nucleic acid molecule, incubation of the reaction mixture for primer extension though the action of a polymerase, contacting the population of nucleic acid molecules in the reaction mixture with a nuclease, purifying duplex nucleic acid molecules comprising the variant nucleic acid molecule. The present disclosure also provides a method of designing a primer.

The present invention relates to a method for DNA strand sequencing, the method comprising (S200) providing a double-stranded DNA fragment having a first strand and a second strand, the DNA strand to be sequenced corresponding to the first strand or the second strand, (S300A) linking the first strand and the second strand with a first hairpin oligonucleotide and a second hairpin oligonucleotide, (S500A) amplifying the ligation product obtained in step (S300A) using rolling circle amplification (RCA); (S600A) amplifying the ligation product obtained in step (S300A) using RCA, or extending the amplicon obtained in step (S500A) using a primer using a first set of redox-modified nucleotides and a second set of redox-modified nucleotides, and (S800A) determining the sequence of the DNA strand by sequencing the amplicon (80) obtained in step (S600A) using an electrochemical sequencing method.

Methods of single molecule sequencing are provided. The primer may be hybridized to a template molecule to form a hybridized template. The sequence of a first region of the template molecule can be determined by extending the primer through the first region using labeled nucleotides. The primer may be extended through a second region of the template using an unlabeled nucleotide. The sequence of a third region of the template molecule can be determined by extending the primer through the third region using a labeled nucleotide. The sequence of the second region of the template molecule may be determined by comparing the sequence of the first region and the sequence of the third region to a reference sequence.

The present disclosure provides methods, systems, and kits for processing nucleic acid molecules. A method may comprise providing a template nucleic acid fragment (e.g., within a cell, cell bead, or cell nucleus) within a partition (e.g., a droplet or well) and subjecting the template nucleic acid fragment to one or more processes including a barcoding process and a single primer extension or amplification process. The processed template nucleic acid fragment may then be recovered from the partition and subjected to further amplification to provide material for subsequent sequencing analysis. The methods provided herein may permit simultaneous processing and analysis of both DNA and RNA molecules originating from the same cell, cell bead, or cell nucleus.

The invention discloses a loop-mediated isothermal amplification (LAMP) detection method of tsRNA, and belongs to the technical field of gene detection. Comprising detection of tsRNA without a ring structure and tsRNA containing the ring structure. The method comprises the following steps: designing a specific primer combination according to requirements, hybridizing tsRNA with Loop A under an isothermal condition, and extending along the primer Loop A under the action of Bst DNA polymerase and dNTPs to form double-stranded DNA; a double-stem-loop structure is released by utilizing the strand displacement activity of Bst DNA polymerase; under the action of the forward inner primer FIP and the reverse inner primer BIP, the double-stem-loop structure is used as a starting template, and LAMP explosive amplification is started; and an amplification product is monitored in real time through a fluorescence signal of the dye, so that quantitative detection of the target tsRNA is realized. The LAMP initial template is formed by detecting the specific hybridization mechanism of the target tsRNA and the primer, so that the target detected tsRNA can directly participate in the LAMP reaction, the pain point that the traditional RT-LAMP method depends on reverse transcriptase and needs target nucleic acid greater than 200nt is avoided, and the rapid tsRNA detection is realized.

This application relates to multiplexed methods for the identification and quantification of minor alleles and polymorphisms. Products and methods are provided for detecting the presence of minor nucleic acid substances in a sample containing a mixture of minor nucleic acid substances and one or more major nucleic acid substances, wherein the amount (frequency or copy number) of the minor nucleic acid substances is less than the amount of the major nucleic acid substances. Some methods involve amplifying the mixture and extending the resulting amplicons with a chain terminator and an extension primer that hybridizes specifically to the amplicons, wherein the chain terminator specific to the major nucleic acid substances has a lower concentration than the chain terminator specific to the minor nucleic acid substances. A higher concentration of the chain terminator specific to the minor nucleic acid substances, relative to the chain terminator specific to the major nucleic acid substances, improves the detection limit (sensitivity) for detecting minor nucleic acid substances present in the mixture at low frequency or copy number. Additionally, the signal generated from the extension product of the major nucleic acid substance amplicons can be used as a positive control and allows for the quantification of the minor nucleic acid substances relative to the major nucleic acid substances in the mixture.

The present disclosure relates to methods and kits for analyzing a macromolecule analyte in a cyclic manner that incorporate basic steps of the ProteoCode™ protein sequencing assay and in addition incorporate approaches that allow to control template independent nucleotide addition by the DNA polymerase, and thus ensure successful multi-cycling encoding. Three different approaches are proposed herein to prevent heterogeneity in the spacer length of the extended recording tag created during the primer extension reaction. The disclosed methods allow for highly-parallelized, accurate and sensitive macromolecule characterization.

This invention discloses a synthetic sequencing method based on nucleotide dimers as monomers. The synthesized nucleotides for sequencing are nucleotide dimers. Under the action of polymerase, if the nucleotide dimer completely pairs with two bases in the DNA template hybridized to the sequencing primer, a polymerization reaction occurs, and the sequencing primer extends by two bases. If the two bases in the DNA template do not completely pair, the polymerization reaction does not occur. This method improves the length and accuracy of sequence determination, enabling high-throughput detection of nucleic acid sequences.

The invention provides methods of detecting genetic features that only require a single successful interrogation. A sample that includes nucleic acid may have a first set of adaptors ligated to free ends in a manner that is not target specific. Then, a reagent such as a Cas endonuclease is used to cut nucleic acid molecules that contain a target of interest, leaving a cut end. A second adaptor type is attached to the cut end(s) and provides a primer binding site. A primer bound to the second adaptor site is extended through the target of interest and through a strand of the first adaptor. This extension yields a template polynucleotide that includes the primer, the copy of the target, and the copy of the first adaptor.

A method including steps of (a) providing an array of sites, wherein each site comprises a mixture of different nucleic acid templates; (b) extending primers hybridized to the different nucleic acid templates at each of the sites with different nucleotide analogs having different reversible blocking moieties, respectively, thereby producing different primer extension products at each site; (c) detecting the different primer extension products to distinguish the different nucleotide analogs at each site; and (d) removing the different reversible blocking moieties from the primer extension products at each of the sites using a first treatment that is selective for a first of the different reversible blocking moieties and a second treatment that is selective for a second of the different reversible blocking moieties.

The present invention relates to: a method for efficiently selecting a high-affinity candidate ligand from a biological sample by using candidate ligands including a DNA barcode and an aptamer specifically binding to a target protein; and a kit therefor. Specifically, a target protein-aptamer-candidate ligand complex is formed by reacting a target protein, a candidate ligand, and an aptamer binding to the target protein, candidate ligands that do not form the complex are removed by nuclease treatment, and then a high-affinity candidate ligand is selected by primer extension and quantitative analysis of a DNA barcode of the candidate ligand bound to the complex. The method according to the present invention can ensure high specificity and reproducibility even in physiological environments such as cell lysates, and can be applied to various protein targets, and thus can be used in a wide range of bio-research fields such as new drug development and diagnostic ligand search.

A method of characterizing a nucleic acid, the method comprising the steps of: (a) contacting a primer-template nucleic acid hybrid with a polymerase and a mixture of nucleotides to produce an extended primer hybrid and form a stable ternary complex comprising the extended primer hybrid, the polymerase, and a nucleotide cognate of a next base in the template, wherein the mixture contains nucleotide cognates of four different base types, wherein the nucleotide cognate of a first base type has a reversible terminator, and wherein nucleotide cognates of second, third, and fourth base types are extendable; (b) detecting the stable ternary complex to distinguish the next base from other base types in the template; and (c) determining the presence of a base multiplet in the template nucleic acid, the base multiplet comprising the first base type followed by the next base.