60 results about "Primer extension" patented technology

Described herein are methods of generating a coupled sequencing read pair for a polynucleotide, and methods of analyzing the coupled sequencing read pair. The coupled sequencing read pair can be analyzed to detect polynucleotide variants, including at loci that are not directly sequenced within the coupled sequencing read pair. Other analytical methods can include using coupled sequencing read pairs to construct or validate a consensus sequence. The coupled sequencing read pair may be generated for a polynucleotide by generating sequencing data for a first region by extending a primer using labeled nucleotides; further extending the primer through a second region using nucleotides provided in a second region flow order, wherein primer extension through the second region is faster than primer extension through the first region; and generating sequencing data associated with a sequence of a third region of the polynucleotide by further extending the primer using labeled nucleotides.

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 invention discloses a method for targeted enrichment of polymorphic sites by introducing thio-modified nucleic acid into a single base. According to the invention, the selector contains a segment of primer extension binding region with more than four basic groups, when the selector is extended by taking a target DNA / RNA as a template, single thio-modified dNTP and another three terminated dNTP / ddNTP, denaturation and renaturation are carried out, a hairpin structure is formed by itself, the selector without thio-modification is digested under the action of T4DNA polymerase, and then the target DNA / RNA is converted into the target DNA / RNA, and the target DNA / RNA is converted into the target DNA / RNA, and then the target DNA / RNA is converted into the target DNA / RNA, and the target DNA / RNA is converted into the target DNA / RNA, and the target DNA / RNA is converted into the target DNA / RNA. And complementing the sequence at the incision to form an intermediate product with a hairpin structure, so as to fulfill the aim of enriching rare mutation sites. The method is suitable for DNA and RNA templates, and the obtained hairpin ring structure intermediate can be subjected to signal amplification in two ways: enzyme-mediated nucleic acid amplification or PCR amplification after linearization of a ring structure.

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 invention discloses a resolution positioning analysis method for an 8-oxo-2 '-deoxyguanine modified single base in DNA (Deoxyribonucleic Acid), and belongs to the technical field of biology. The method comprises the following steps: firstly, extracting genome DNA, ultrasonically breaking, then denaturing into single-stranded DNA, and enriching the single-stranded DNA containing 8-oxo-dG by using an antibody; adding a 3 ' / 5' universal sequencing joint to the enriched single-stranded DNA; fixing the amino-modified extension primer to the carboxyl-modified magnetic bead to obtain an extension primer fixed by the carboxyl-modified magnetic bead; extension is carried out through an extension primer fixed by a carboxyl modified magnetic bead, and 8-oxo-2'deoxyguanine-thymine in DNA polymerase mediation enrichment single-stranded DNA is converted, so that a primer extension product fixed on the magnetic bead is obtained; purifying a primer extension product fixed on the magnetic beads; and carrying out PCR amplification on the primer extension product, and carrying out enzyme digestion or sequencing analysis. According to the method, the reaction system is mild, the defect that DNA oxidation modification is possibly artificially introduced in an existing chemical labeling method is avoided, and Ggt is passed; t conversion is carried out, so that the 8-oxo-dG can be modified, positioned and analyzed more directly and accurately.

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.

The invention discloses a method for generating a hairpin ring for enriching mutation or allele information. According to the invention, a primer extension binding region containing more than 8 basic groups is arranged on a selector; when the selector extends by taking the target DNA as a template and is denatured and renatured, a 3 '-terminal suspended hairpin structure is formed by itself, a 3'-terminal suspended sequence is digested under the action of T4DNA polymerase, a sequence at a gap is supplemented, a hairpin structure intermediate product is formed, and the hairpin structure is used for synthesizing a DNA sequence by taking a genome as a template. And carrying out targeted enrichment on polymorphic site information (wild type and mutant type), and then carrying out information decoding through next-generation sequencing. The method is used for enriching information of rare mutation sites, only common dNTP is needed for single primer amplification, then T4DNA polymerase is added for digesting mismatched sequences in a stem region, completely paired sequences are reserved, and the purpose of enriching the rare mutation sites is achieved. And more than 1000 probes can be subjected to multiple analysis in one PCR tube by using standard laboratory equipment.

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.

A method for analyzing planar sample is provided. In some cases the method comprises: (a) labelling the planar sample with a capture agent that is linked to a nucleic acid, wherein the capture agent specifically binds to complementary sites in the planar sample; (b) reading a fluorescent signal caused by extension of a primer that is hybridized to the nucleic acid, using fluorescence microscopy. Several implementations of the method, and multiplexed versions of the same, are also provided.

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.

Methods for generating paired sequencing reads for a polynucleotide, and methods for analyzing paired sequencing reads, are described. Paired sequencing reads can be analyzed to detect polynucleotide variants, including at loci that are not directly sequenced within the paired sequencing reads. Other methods of analysis include using paired sequencing reads to construct or validate a consensus sequence. Paired sequencing reads for a polynucleotide can be generated as follows: by extending a primer with a labeled nucleotide to generate sequencing data for a first region; further extending the primer through a second region using nucleotides provided in a second region flow order, wherein the primer extends through the second region faster than the primer extends through the first region; and generating sequencing data related to the sequence of a third region of the polynucleotide by further extending the primer with a labeled nucleotide.

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 invention relates to the technical field of forensic medicine identification, in particular to a genetic marker system containing 91 high-performance autosomal microhaplotypes as well as a detection primer and application of the genetic marker system. The invention provides a genetic marker system containing 91 high-performance autosomal microhaplotypes. According to the system, a single-ended primer extension technology is utilized for amplification, and the requirement for the integrity of DNA fragments is lower. The specific application of single-ended primer extension in the system is as follows: at least one specific primer is designed for each micro haplotype site, and the other end is a universal primer suitable for all sites in the composite system. The universal primer is combined at the fracture position of the target fragment, so that even if the target DNA fragment is fractured, partial amplification can still be performed and sequencing can be completed.

The present invention is a method and compositions for primer extension target enrichment of nucleic acids and improvements thereto including simultaneously enriching for RNA and DNA and optionally sequencing the enriched products.

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.

This disclosure provides a technology for improving the length and accuracy of sequence reads obtained from a DNA fragment library. Amplicons (such as DNA nanoballs or PCR clusters) are prepared that have longer fragment inserts. This makes the amplicons larger, resulting in lower signal intensity per amplicon, and increasing the likelihood that primer extension products will go out of phase. To compensate, the amplicons are arrayed closer together, and compacted using crosslinking oligonucleotides and a high magnesium concentration. Primer extension products are kept in phase by improving incorporation of bases, ensuring that reactions go to completion, and purging unused reagents. Data collection is improved with brighter detection reagents using a two color four image (2c4i) detection protocol. When these features are combined, accurate reads of 600 to 1000 base pairs or more are routinely obtained.

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 pertains to compounds, methods and compositions for improving on nucleic acid polymerization, including DNA replication by in vitro primer extension to generate, for example, polymers for nanopore-based single molecule sequencing of a DNA template. A nucleic acid polymerase reaction composition is provided with polymerization enhancement moieties, which allows enhanced DNA polymerase activity with nucleotide analogs, resulting in improved length of primer extension products for sequencing applications.

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.

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.

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.