46 results about "Concatemer" patented technology

The present disclosure provides methods for reducing sequencing errors comprising one or any combination of: (i) removing deaminated bases in any nucleic acid molecule throughout a library preparation workflow which includes immobilised splints which bind to the library, the use of a compaction oligonucleotide, optionally with an intervening sequence, formation of closed circular nucleic acids, creating gaps using glycosylase and lyase activities at positions with deaminated bases. The library may be sequenced using pairwise sequencing, e.g. with dark sequencing and / or sequencing using a multivalent labelled probe for the formation of an avidity molecule and soluble primer and polymerase. Method for sequencing concatemers in which the concatermers are contacted with polymerases, soluble primers and a multivalent labelled molecule which forms a complex with the polymerase. Detecting polymerase position and nucleobase bound to the polymerase in the complex. These methods generate higher quality base calls during downstream sequencing workflows.

The present disclosure provides compositions, apparatus and methods for generating a plurality of concatemer template molecules immobilized on a support for conducting massively parallel sequencing runs. In some embodiments, the concatemer template molecules can be generated by conducting rolling circle amplification reactions on a support comprising a mixture of immobilized capture and pinning primers. The rolling circle amplification reaction comprises a plurality of circularized polynucleotide molecules and soluble amplification primers which generates concatemer template molecules that collapse to form compact DNA nanoballs that are stably immobilized to a support.

The present disclosure provides compositions and methods that employ the compositions for conducting pairwise sequencing and for generating concatemer template molecules for pairwise sequencing. The concatemers can be generated using a rolling circle amplification reaction which is conducted either on-support, or conducted in-solution and then distributed onto a support. The rolling circle amplification reaction generates concatemers containing tandem copies of a sequence of interest and at least one universal adaptor sequence. An increase in the number of tandem copies in a given concatemer increases the number of sites along the concatemer for hybridizing to multiple sequencing primers which serve as multiple initiation sites for polymerase-catalyzed sequencing reactions. When the sequencing reaction employs detectably labeled nucleotides and / or detectably labeled multivalent molecules (e.g., having nucleotide units), the signals emitted by the nucleotides or nucleotide units that participate in the parallel sequencing reactions along the concatemer yields an increased signal intensity for each concatemer.

The present disclosure provides compositions and related methods, e.g., for preparing immobilized nucleic acid nanostructures using compaction oligonucleotides. In some embodiments, rolling circle amplification reaction can be conducted with compaction oligonucleotides on-support or in-solution to generate concatemer molecules having multiple copies of a polynucleotide unit arranged in tandem. Each polynucleotide unit comprises a sequence-of-interest and at least one universal adaptor sequence that binds one end of a compaction oligonucleotide. The 5′ and 3′ regions of the compaction oligonucleotide can hybridize to the concatemer to pull together distal portions of the concatemer causing compaction of the concatemer to form a nanostructure. Nanostructures having tighter size and shape compared to concatemers generated in the absence of the compaction oligonucleotides. The compact and stable characteristics of the nucleic acid nanostructures improves sequencing accuracy by increasing signal intensity and they retain their shape and size during multiple sequencing cycles.

The present disclosure provides engineered bacterial cells having one or more genetic modifications that result in increased production of expression products, such as increased production of plasmids with reduced occurrence of plasmid concatemers or multimers. Also disclosed herein are expression systems for product manufacturing in media having reduced antibiotic concentration, as well as methods of making cells for use in the expression systems. The cells and expression systems increase the growth rate and production yields of biomolecules produced within the host bacterial host cells without the use of antibiotics, and can produce the product with little to no endotoxins present.

The present disclosure provides engineered bacterial cells having one or more genetic modifications that result in increased production of expression products, such as increased production of plasmids with reduced occurrence of plasmid concatemers or multimers. Also disclosed herein are expression systems for product manufacturing in media having reduced antibiotic concentration, as well as methods of making cells for use in the expression systems. The cells and expression systems increase the growth rate and production yields of biomolecules produced within the host bacterial host cells without the use of antibiotics, and can produce the product with little to no endotoxins present.

Provided are techniques for generating expression products using one or more nucleic acid concatemers that include tandem repeats of a nucleic acid sequence encoding the expression product or products. In one embodiment, different expression products may be co-expressed using a concatemer mixture of a first nucleic acid concatemer and a second nucleic acid concatemer having a predefined ratio to one another.

The present invention relates nucleic acid molecules and concatemers containing spacer sequences useful for the efficient packaging of viral particles so as to minimize the incorporation of contaminant nucleic acids into these vectors, as well as methods of producing such viral particles.

The invention provides an application of a GPRC5D humanized antibody and a concatemer in construction of a CAR-T cell, particularly relates to a GPRC5D-targeted single-domain antibody and the humanized antibody thereof, and a serial configuration of the antibody, and also provides a chimeric antigen receptor (CAR) molecule constructed by using the antibody, the CAR-T cell and application thereof. Under a standardized condition, the tandem configuration shows remarkably improved tumor cell killing compared with a corresponding monomer configuration so as to realize a high CAR-T maximum effect, and a new treatment choice is provided for GPRC5D positive multiple myeloma patients.

The present disclosure provides a universal mRNA tumor vaccine and a preparation method and use thereof. The universal mRNA tumor vaccine is constructed based on a concatemer antigen peptide, and comprises a combined peptide segment containing at least two tumor-specific antigen peptides and tumor-associated antigen peptides. The concatemer antigen peptide provided by the present disclosure comprises a plurality of immunogenic antigens. The mRNA tumor vaccine constructed based thereon has good antigen expression efficiency in cells and can produce strong cellular immune effects, and can be used for treating and / or preventing tumors (e.g., colorectal cancer), and can also be combined with other anti-tumor drugs (e.g., anti-PD-1 antibodies) for treating tumors (e.g., colorectal cancer).

The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address one or more issues associated with the stability and / or size of nucleic acid structures, such as RCPs, in the biological sample without the use of exogenously added oligonucleotide compaction probes. In some embodiments, provided herein are methods involving the use of self-hybridizing hybridizing regions for compacting and / or stabilizing nucleic acid concatemers (e.g., RCPs). In some embodiments, dynamic inter-strand annealing between tandem units of an RCP is used for compaction and / or stabilization. In some embodiments, short palindromic regions in an RCP are used for compaction and / or stabilization.

Provided are methods and compositions used for paired-end sequencing. The method comprises providing a DNA array comprising a surface immobilized with DNA concatemers. Foreach of the plurality of DNA concatemers on the array, the method comprises annealing first read primers to primer binding sites on the DNA concatemer, extending at least some of the first read primers to incorporate dNTPs or dNTP analogs, thereby producing first read strands, each of the dNTPs or dNTP analogs being incorporated is identified to produce first reads, performing controlled MDA by extending at least some of the first read strands with a polymerase having strand-displacement activity to generate a plurallty of second strands, each second strand comprising a portion that is hybridized to the DNA concatemer, and an unhybridized, single-stranded branch; and annealing second read primers to the single-stranded branches of the plurality of second strands, and extending the second read primers to generate the second reads.

The invention relates to a method and a kit for generating nucleic acid for target capture. The method involves binding of a circ-olio sequence to at least one target oligonucleotide to generate a single stranded circular DNA. The single stranded circular DNA is bidirectionally amplified with the sense and antisense oligonucleotides, followed by biotinylation to obtain for a double-stranded concatemer of linear sequence. The double-stranded concatemer of linear sequence with the restriction endonucleases produces a target capture nucleic acid. The method offers a scalable targeted capture of pr eselected genomic regions, and is cost effective. The method of generating nucleic acid for target capture has application in the field of oncology hematology, inherited malignancies, germline disorders, microbiology and prenatal diagnosis. The invention generates baits over high GC-rich regions of the genome.

The present disclosure provides compositions and methods for preparing a plurality of linear nucleic acid library molecules. In some embodiments, two or more of the library preparation steps can be conducted in a single reaction vessel (e.g., one-pot library prep workflow) resulting in a streamlined workflow that requires reduced hands-on manipulation and time. In some embodiments, the plurality of linear library molecules can be circularized using soluble or immobilized splint oligonucleotides. In some embodiments, the circular library molecules can be subjected to rolling circle amplification and the resulting concatemer template molecules can be sequenced.

The present invention provides a variant pseudorabies virus (PRV) gC-gD epitope concatemer and its application. The amino acid sequence of the variant pseudorabies virus gC-gD expression concatemer is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2. The present invention uses the variant pseudorabies virus gC-gD epitope concatemer to establish an indirect ELISA detection method for PRV neutralizing antibodies with strong specificity, high sensitivity and good repeatability. The method can fully meet the serological diagnosis requirements of pig herds in pig farms where PRV wild virus is prevalent and pig farms where PRV wild virus has been purified, laying a certain foundation for the development of a new PRV polypeptide vaccine.

The present disclosure in some aspects relates to methods and compositions for accurately detecting and quantifying multiple analytes present in a biological sample. In some aspects, the methods and compositions provided herein address one or more issues associated with the stability and / or size of nucleic acid structures, such as RCPs, in the biological sample without the use of exogenously added oligonucleotide compaction probes. In some embodiments, provided herein are methods involving the use of self-hybridizing hybridizing regions for compacting and / or stabilizing nucleic acid concatemers (e.g., RCPs). In some embodiments, dynamic inter-strand annealing between tandem units of an RCP is used for compaction and / or stabilization. In some embodiments, short palindromic regions in an RCP are used for compaction and / or stabilization.

Random arrays of single molecules are provided for carrying out large scale analyzes, particularly of biomolecules, such as genomic DNA, cDNAs, proteins, and the like. In one aspect, arrays of the invention comprise concatemers of DNA fragments that are randomly disposed on a regular array of discrete spaced apart regions, such that substantially all such regions contain no more than a single concatemer. Preferably, such regions have areas substantially less than 1 μm2 and have nearest neighbor distances that permit optical resolution of on the order of 109 single molecules per cm2. Many analytical chemistries can be applied to random arrays of the invention, including sequencing by hybridization chemistries, sequencing by synthesis chemistries, SNP detection chemistries, and the like, to greatly expand the scale and potential applications of such techniques.

The present application relates to methods and compositions for dual - strand sequencing. The method includes providing a DNA array comprising a surface to which DNA concatemers are immobilized. For each of a plurality of DNA concatemers on the array, the method includes annealing a first read primer to a primer binding site on the DNA concatemer, extending at least some of the first read primer to incorporate dNTPs or dNTP analogs, thereby generating a first read strand, wherein each incorporated dNTP or dNTP analog is identified as generating a first read, performing controlled MDA by extending at least some of the first read strand with a polymerase having strand displacement activity to generate multiple second strands, each second strand comprising a portion hybridized to the DNA concatemer and an unhybridized single - stranded branch; and annealing a second read primer to the single - stranded branches of the multiple second strands and extending the second read primer to generate a second read.

This application relates to methods and compositions used for paired-end sequencing. The method comprises providing a DNA array comprising a surface immobilized with DNA concatemers. For each of the plurality of DNA concatemers on the array, the method comprises annealing first read primers to primer binding sites on the DNA concatemer, extending at least some of the first read primers to incorporate dNTPs or dNTP analogs, thereby producing first read strands. Each of the dNTPs or dNTP analogs being incorporated is identified to produce first reads, performing controlled MDA by extending at least some of the first read strands with a polymerase having strand-displacement activity to generate a plurality of partially hybridized second strands. Second read primers are then annealed to the single-stranded branches of the plurality of second strands to generate the second reads.

Provided herein are compositions and methods for spatial profiling of biomolecules in a sample. Methods comprise photocrosslinked oligo-mediated cleavage (POMC), utilizing molecular proximity recording to generating spatial inference data without the need for indexing probe locations. Processes include disclosed herein generating concatemers of docking oligonucleotides, photo crosslinking oligonucleotides to generate cleavage sites, and generating spatial information from generated fragments.

The invention discloses a construction method and application of an AAV single-molecule sequencing library and a kit, and belongs to the technical field of biological medicine detection. In order to solve the technical problem that ligase steric hindrance is caused by a secondary structure of an AAV inverted repeat (ITR), a Cas9-RNP compound specifically targeting an ITR stem region (Stem Region) is used for carrying out directional cutting on a single-chain genome, a closed-loop hairpin structure is removed, and a blunt-end double-chain DNA handle of 10-50 bp is reserved at the tail end. In cooperation with a forced dissociation step of the enzyme, the method eliminates connection steric hindrance and significantly improves the linker connection efficiency. According to the method, double-end cyclization is not needed, sequencing can be achieved only through single-end connection, and therefore a complete genome, a truncated body and a covalent head-to-tail concatemer can be captured without bias, and the method can be used for accurately measuring the physical packaging capacity limit of the AAV carrier.

A method for determining a sequence from a sense strand and an antisense strand of a nucleic acid, comprising (a) providing a cluster of nucleic acids attached to a solid support, wherein the cluster of nucleic acids comprises a sense strand and an antisense strand of a concatemer, the concatemer comprising a plurality of copies of a sequence unit, the sequence unit comprising a target sequence and a primer binding site; (b) hybridizing a primer to the primer binding site in the antisense strand; (c) extending the primer along the antisense strand to determine a sequence from at least a portion of the target sequence in the antisense strand; (d) hybridizing a second primer to the primer binding site in the sense strand; and (e) extending the second primer along the sense strand to determine a sequence from at least a portion of the target sequence in the sense strand.

The invention relates to a method for preparing scDNA, which comprises the following steps: providing a circular nucleic acid template which is double-stranded and comprises a to-be-recombined ring-forming region and a to-be-enzyme-digested incision site arranged outside the region, and the to-be-recombined ring-forming region comprises an expression framework and a recombination site pair arranged on two wings of the expression framework; the present invention relates to a method for preparing a cyclic nucleic acid template, the cyclic nucleic acid template being amplified by RCA rolling circle amplification to form a concatemer, the concatemer comprising a tandem repeat unit of a cyclic nucleic acid template sequence, the concatemer being linearized by enzyme digestion in the incision site of the tandem repeat unit with a restriction enzyme simultaneously with or subsequent to the rolling circle amplification, the linearized concatemer comprising a cyclic nucleic acid template sequence, the cyclic nucleic acid template comprising a cyclic nucleic acid template sequence, and the cyclic nucleic acid template sequence comprising a cyclic nucleic acid template sequence. Free linearized nucleic acid is obtained; in a cell-free system, the linearized nucleic acid is recombined with a recombinase into scDNA comprising the expression framework. The method provided by the invention really realizes efficient preparation of scDNA.

Provided are methods of nucleic acid sequencing. The methods include producing a circularized DNA including a full-length cDNA and a known heterologous sequence, and performing rolling circle amplification using the circularized DNA as template to produce a concatemer including repeating segments including the full-length cDNA and the known heterologous sequence. The methods further include obtaining a raw sequencing read of the concatemer using a nanopore, identifying the repeating segments in the raw sequencing read, and producing a consensus sequence of the full-length cDNA based on the sequences of the repeating segments. Computer-readable media, computing devices, and systems that find use, e.g., in practicing the methods of the present disclosure are also provided.

The application discloses a kit and a method for detecting trace protein. The kit in the application can detect MMP13 and SCC protein at a level as low as fg / mL based on DNA string combination technology and a CRISPR-Cas12a system, and has high sensitivity. Moreover, the kit can simultaneously get rid of dependence on real-time amplification reaction, has good stability, can effectively avoid cross contamination between samples, and does not need real-time amplification in the detection process. The used ssDNA string can be prepared in advance, the kit gets rid of the real-time cycle amplification reaction which is dependent on the current detection technology, has low background signal, high stability, and can effectively prevent cross contamination between samples. Moreover, the kit has low cost, does not need to use complex and precise equipment or catalytic enzymes with special functions, and has high universality, and can be popularized to quantitative detection of any kind of protein by only replacing capture antibodies and detection antibodies.

Provided herein are compositions and methods for spatial profiling of biomolecules in a sample. Methods comprise photocrosslinked oligo-mediated cleavage (POMC), utilizing molecular proximity recording to generating spatial inference data without the need for indexing probe locations. Processes include disclosed herein generating concatemers of docking oligonucleotides, photo crosslinking oligonucleotides to generate cleavage sites, and generating spatial information from generated fragments.

The present disclosure provides methods for reducing sequencing errors comprising one or any combination of: (i) removing deaminated bases in any nucleic acid molecule throughout a library preparation workflow which includes immobilised splints which bind to the library, the use of a compaction oligonucleotide, optionally with an intervening sequence, formation of closed circular nucleic acids, creating gaps using glycosylase and lyase activities at positions with deaminated bases. The library may be sequenced using pairwise sequencing, e.g. with dark sequencing and / or sequencing using a multivalent labelled probe for the formation of an avidity molecule and soluble primer and polymerase. Method for sequencing concatemers in which the concatermers are contacted with polymerases, soluble primers and a multivalent labelled molecule which forms a complex with the polymerase. Detecting polymerase position and nucleobase bound to the polymerase in the complex. These methods generate higher quality base calls during downstream sequencing workflows.

A method for determining sequences from sense and antisense strands of a nucleic acid, including (a) providing a nucleic acid cluster attached to a solid support, wherein the nucleic acid cluster includes a sense strand and an antisense strand of a concatemer, the concatemer including multiple copies of a sequence unit, the sequence unit including a target sequence and a primer binding site; (b) hybridizing a primer to a primer binding site in the antisense strand; (c) extending the primer along the antisense strand to determine the sequence from at least a portion of the target sequence in the antisense strand; (d) hybridizing a second primer to a primer binding site in the sense strand; and (e) extending the second primer along the sense strand to determine the sequence from at least a portion of the target sequence in the sense strand.

Provided herein are systems and methods for amplifying nucleic acid samples, immobilizing such amplified products onto substrates, and sequencing the amplified products on the substrates. The amplified products may comprise concatemers.