31 results about "Primer binding site" patented technology

The present invention relates to methods of detecting region(s) of interest in a gene comprising a polyA tail. The region(s) of interest can include gene(s), region(s), mutation(s), deletion(s), insertion(s), indel(s), and / or translocation(s). The region(s) can be greater than or less than 1 kilobases from the polyA tail. Methods can include forming a library of single cell transcripts comprising the region(s) in close proximity to a cell barcode and a unique molecular identifier (UMI). Methods for distinguishing cells by genotype can include amplifying the transcripts using PCR methods and detecting the cell barcode and UMI using single cell sequencing methods. Transcripts can be enriched using tagged region-specific PCR primers. Cell barcodes can be brought into close proximity to the region(s) by circularizing the transcripts. Sequencing of the transcripts can include using primer binding sites added during PCR amplification and library indexes for multiplexed sequencing.

This application discloses a kit and method for quantitative detection of double-stranded DNA libraries compatible with multiple sequencing platforms, relating to the field of sequencing technology. The kit includes standards and a mixed primer combination. The standards include a set of serially diluted double-stranded DNA fragments of known concentration, each fragment including a PCR primer binding site sequence of a sequencing adapter from a first sequencing platform or a PCR primer binding site sequence of a paired-end barcode sequencing adapter from a second sequencing platform. The mixed primer combination includes a first primer pair and a second primer pair. The first primer pair specifically binds to the PCR primer binding region in the sequencing adapter from the first sequencing platform, and the second primer pair specifically binds to the PCR primer binding region in the paired-end barcode sequencing adapter from the second sequencing platform. The molar ratio of the first primer pair to the second primer pair in the mixed primer combination is 1:1. This kit can perform quantitative detection of libraries from different sequencing platforms. All samples are calculated using the same standard curve run, reducing systematic errors.

This invention discloses a method for constructing a multi-target amplicon abundance standard for microorganisms and its application, belonging to the fields of molecular biology detection and microbiome analysis. This standard material screens sequences of common human gut microbiota strains, designs and adds universal primers, and obtains 14 DNA fragments of different lengths through gene synthesis, cloning, and purification, which are then mixed according to a preset abundance. It retains natural characteristics such as primer binding sites and GC content, and can systematically correct technical deviations in amplicon sequencing, solving the problem of species abundance distortion. This standard material functions as both a non-homologous internal reference and a homologous external reference, and can be used for laboratory proficiency testing, reagent kit performance evaluation, and cross-platform data calibration, promoting the leap from relative qualitative to absolute quantitative research in microbiome studies and providing metrological support for the standardization and precision of detection results.

Provided is a prime editing guide RNA (pegRNA), aprime editing (PE) system, and use thereof, pertaining to the technical field of prime editing. The prime editing guide RNA provided herein positions the reverse transcriptase template and primer binding site (RTT-PBS) at the stem loop 2 region of the sgRNA scaffold based on the canonical pegRNA design. This configuration not only effectively mitigates degradation of the RTT-PBS but also increases its binding capability to Cas9. Consequently, this design preserves the efficiency of prime editing without interference.

The present invention relates to the field of genomic engineering. In particular, a chimeric prime editing (cPE) system is disclosed comprising elements including, but not limited to a Cas9 nickase (nCas9) / high fidelity nucleotide polymerase (HFNTPol) RNA, one or more single guide RNAs (sgRNAs), and a chimeric prime editor template oligonucleotide (cpetODN) comprising a deoxyribonucleic acid nucleotide polymerase template (NPT) and a primer binding site. For example, the sgRNA and the cpetODN are ligated into a single oligonucleotide. Alternatively, the sgRNA and the cpetODN are free and independent molecules (e.g., modular). This cPE system results in precise and efficient genome editing in cells and in adult mouse liver which is advantageous over conventional sgRNA prime editor fusion constructs. This flexible and modular system is an improvement in the art to obtain precise genome editing.

In one aspect, the disclosure relates to a system for site-specific modification of a double­stranded target DNA sequence, the system including at least: a fusion protein comprising an RNA-binding nickase protein and a DNA-dependent DNA polymerase, and a chimeric guide nucleic acid sequence comprising a guide RNA sequence that interacts with the RNA-binding nickase protein and a single-stranded DNA template, wherein the single-stranded DNA template comprises a modified sequence for insertion into a second strand of the double-stranded target DNA sequence and a primer binding site. Also disclosed is a method for site-specific modification of a double-stranded target DNA sequence in a cell. In another aspect, the present disclosure is directed to a fusion protein including an RNA-binding nickase protein and a DNA-dependent DNA polymerase, wherein one or both of the nicakse protein and the DNA polymerase can include one or more mutations to optimize function and prevent off-target effects.

Described herein are methods for amplifying template / target nucleic acids by employing long primers, where the original template / target nucleic acid has short primer binding sites (e.g., < = 15 nucleotides). The long forward primer and the long reverse primer have the following two regions: (1) a region for annealing / hybridizing with a region of the template / target nucleic acid; and (2) a region that is not annealed / hybridized to the region of the template / target nucleic acid. In other words, the long primer is longer than the primer binding site (i.e., the site at which the long primer is to anneal / hybridize). The results of using these long primers overcome the problems and obstacles of short primer binding sequences.

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 present invention relates to a novel retroviral vector comprising a 5'long terminal repeat (LTR), a primer binding site, a heterologous intron, a transgene insertion site and a 3 'LTR wherein the heterologous intron is located immediately downstream of the 5' LTR. This vector has high titer productivity and is particularly suitable for gene therapy.

The invention relates to the field of gene engineering, and discloses a pilot editing system, a recombinant strain containing the pilot editing system, application of the recombinant strain and a gene editing method. The system comprises a coding gene of nicking enzyme nCas9, a coding gene of reverse transcriptase RT and pegRNA. Wherein the coding gene of the nicking enzyme nCas9 and the coding gene of the reverse transcriptase RT are connected through a Linker sequence, and the nucleotide sequence of the Linker sequence is as shown in SEQ ID NO. 1; the pegRNA comprises a gibberella zeylanica endogenous promoter 5SrRNA, sgRNA, a spacer sequence spacer, a primer binding site PBS (Phosphate Buffer Solution) and a reverse transcription template RT template; the reverse transcription template RT template carries an insertion sequence, and the insertion sequence is used for destroying the expression of a target gene. The method realizes efficient, accurate and safe gene editing of gibberella zeylanica, and has important scientific research and application values.

The technology described herein relates to circularized prime editing guide RNAs (cpegRNAs) comprising at least a spacer, a gRNA scaffold, a primer binding site, and a template sequence with one or more nucleotide changes relative to a target sequence. The disclosure also provides compositions and prime editing systems comprising the pegRNAs and uses thereof for prime editing.

The invention discloses a multi-modal nucleic acid detection method (multi-modal LAMP, M-LAMP) of an LAMP (loop-mediated isothermal amplification) auxiliary molecular beacon system based on connection start. In M-LAMP, aiming at different targets to be detected, only target binding sites can be changed, and primer binding sites and signal probe binding sites are fixed, so that single, double and triple detection can be simultaneously realized by using one set of LAMP primer and signal probe, and the tedious and time-consuming design and optimization process of the primer and signal probe is avoided. The two major problems, namely the primer design and optimization complexity and the false positive risk, existing in the current carbapenemase detection based on LAMP are effectively solved, and the reliability and timeliness of a detection result are remarkably improved.

Provided are prime editing systems with modification-tolerant polymerases and petRNAs and / or pegRNA with chemically modified primer binding sites (PBS) and / or nucleotide polymerase template (NPT).

Provided are prime editing systems with modification-tolerant polymerases and petRNAs and / or pegRNA with chemically modified primer binding sites (PBS) and / or nucleotide polymerase template (NPT).

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.

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 control compositions for a quantitative polymerase chain reaction. More particularly, the invention relates to control compositions for a quantitative polymerase chain reaction having at least one barcode sequence fragment and at least a first and a second primer binding site fragment, and to methods of their use.

Provided herein are engineered split prime editors (PEs) and optimized pegRNAs (with optimized reverse transcriptase template (RTT) length, primer binding site (PBS) length, engineered pegRNA (epegRNA) composition, silent mutation type and location, RNA linkers, and RT initiation sequences) and their use in correcting a prevalent deafness-causing mutation, GJB2 c35delG.

The invention discloses an editing guiding system and application thereof in plant genome editing. The guided editing system comprises a fusion protein, a substance for reducing the content and / or activity of RDR6 protein, sgRNA and pegRNA, the fusion protein is formed by fusing nuclease and reverse transcriptase; the sgRNA is used for generating a non-editing strand incision; the pegRNA comprises sgRNA ', a reverse transcription template sequence and a primer binding site sequence, and the sgRNA' is used for editing a chain target sequence in a targeted manner. The guiding editing system provided by the invention breaks through the application bottleneck of a PE system in arabidopsis thaliana, greatly improves the PE editing efficiency of an arabidopsis thaliana target, and provides a more accurate and powerful tool for arabidopsis thaliana gene function research.

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.

The invention relates to methods and compositions for double-terminal sequencing. The method comprises providing a DNA array comprising a surface on which DNA concatemers are immobilized. For each of a plurality of DNA concatemers on the array, the method includes annealing a first reading primer to a primer binding site on the DNA concatemer, extending at least some of the first reading primer to incorporate dNTP or dNTP analogs, thereby producing a first reading strand, where each incorporated dNTP or dNTP analog is identified as producing a first read segment, and extending at least some of the first reading primer to incorporate dNTP or dNTP analog to produce a second read segment. Performing controlled MDA by extending at least some of the first read strands with a polymerase having strand displacement activity to produce a plurality of second strands, each second strand comprising a moiety that hybridizes to the DNA concatemer and an unhybridized single strand branch; and annealing the second reading primer to single chain branches of the plurality of second chains, and extending the second reading primer to produce a second read.

The present invention belongs to the technical field of plant germplasm resource identification, and specifically relates to a CDDP molecular marker primer set for identifying Polygonatum germplasm resources and its application. The CDDP molecular marker primer set is designed for WRKY, MYB, ERF, KNOX and MADS. The present invention designs primers using the conserved gene sequences corresponding to the five conserved amino acid sequences of WRKY, MYB, ERF, KNOX and MADS as reference sequences. These short conserved gene sequences exist at multiple locations in the plant genome and provide multiple primer binding sites. This type of primer design focuses on gene regions and has advantages over random markers. At the same time, the designed primers are used to amplify genomic DNA of Polygonatum germplasm, which can be used for the identification, differentiation and genetic diversity analysis of Polygonatum germplasm resources.

The invention provides a gene editing system Myc-G4 pegRNA construction method based on a Myc-G4 chain body, which comprises the following steps: designing an eomesa pegRNA target site sequence according to a No.1 exon sequence of an eomesa gene, and selecting a reverse transcription template and a primer binding site sequence length to design a reverse primer sequence; the method comprises the following steps: synthesizing an eomesa pegRNA target site sequence, a reverse primer sequence and a Myc-G4 pegRNA scaffold sequence, and carrying out PCR (Polymerase Chain Reaction) amplification to obtain a Myc-G4 pegRNA template; the gene editing system Myc-G4 pegRNA based on the Myc-G4 chain body is obtained after the Myc-G4 pegRNA template is subjected to purification and recovery, in-vitro transcription and secondary purification and recovery. The invention also provides an application of the constructed Myc-G4 pegRNA, and the constructed Myc-G4 pegRNA is used for editing an eomesa gene for the development of the odd fins of cyprinid fishes. According to the method, the pegRNA structure is optimized through the Myc-G4 chain body, breakthrough improvement of cyprinid fish gene editing efficiency is achieved, meanwhile, the experimental process is simplified, the cost is reduced, and high efficiency, accuracy, convenience and applicability are achieved.

The invention relates to methods and compositions for double-terminal sequencing. The method comprises providing a DNA array comprising a surface on which DNA concatemers are immobilized. For each of a plurality of DNA concatemers on the array, the method includes annealing a first reading primer to a primer binding site on the DNA concatemer, extending at least some of the first reading primer to incorporate dNTP or dNTP analogs, thereby producing a first reading strand, where each incorporated dNTP or dNTP analog is identified as producing a first read segment, and extending at least some of the first reading primer to incorporate dNTP or dNTP analog to produce a second read segment. Performing controlled MDA by extending at least some of the first read strands with a polymerase having strand displacement activity to produce a plurality of second strands, each second strand comprising a moiety that hybridizes to the DNA concatemer and an unhybridized single strand branch; and annealing the second reading primer to single chain branches of the plurality of second chains, and extending the second reading primer to produce a second read.

The technology described herein relates to circularized prime editing guide RNAs (cpegRNAs) comprising at least a spacer, a gRNA scaffold, a primer binding site, and a template sequence with one or more nucleotide changes relative to a target sequence. The disclosure also provides compositions and prime editing systems comprising the pegRNAs and uses thereof for prime editing.

The present invention relates to the field of biotechnology, genetic engineering and medicine, in particular, to a highly efficient system for editing the F508del mutation in the cystic fibrosis transmembrane regulator (CFTR) gene to the wild type, comprising a polynucleotide which comprises a nucleotide sequence, encoding the SpCas9 nickase recognizing PAM NGG or NG, and a prime editing guide RNA (pegRNA) comprising in its structure a sequence complementary to the target locus for editing the F508del mutation in human CFTR gene, a reverse transcription template (RTT) and a primer binding site (PBS), wherein the prime editing guide RNA (pegRNA) has sequence SEQ ID NO: 1-6 or a sequence comprising one or more replacements in the RTT compared to SEQ ID NO: 1-6, selected from SEQ ID NO: 7-168. The system for editing the F508del mutation in the cystic fibrosis transmembrane regulator (CFTR) gene to the wild type provides more efficient editing of the F508del mutation in the cystic fibrosis transmembrane regulator (CFTR) gene to the wild type and is a highly efficient cystic fibrosis treatment. The present invention also relates to the method of editing the F508del mutation in the cystic fibrosis transmembrane regulator (CFTR) gene using the system for editing the F508del mutation in the cystic fibrosis transmembrane regulator (CFTR) gene according to the present invention and the use of the system for editing the F508del mutation in the cystic fibrosis transmembrane regulator (CFTR) gene for treating cystic fibrosis.

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.

The present invention relates to the field of ligation of oligonucleotides to DNA fragments. The ligation methods of the invention may be used for attaching oligonucleotides comprising for example adaptors, primer binding sites, promoters, tags, barcodes or any combination of the aforementioned to DNA fragments.

This invention discloses the development and application of a specific marker associated with the shell thickness trait of the Chinese mitten crab (Eriocheir sinensis). This molecular marker is located in Chr09 of the Chinese mitten crab genome. Esine02654 The gene region exhibits significant sequence length polymorphism: the wild type contains a 72 bp nucleotide sequence, while in the mutant, this sequence is mutated to a single base A or deleted. This invention utilizes the specific design of downstream primers located within this variant region to develop a primer pair for detecting this structural variation. Amplification using this primer pair shows a specific 109 bp band in thick-shelled individuals (wild-type), while thin-shelled individuals (homozygous deletion mutant) show no amplification band due to the absence of the primer binding site. This marker detection is simple, and the results are intuitively interpreted, showing either a band or no band, making it suitable for early-stage auxiliary screening and breeding of the thick-shell trait in Chinese mitten crabs.

The invention is directed to a method for obtaining a nucleic acid library of a sample comprising polynucleotides comprising the steps: a. Providing a plurality of modified primer to the polynucleotides, wherein said modified primer is a starting point for a polymerase for nucleic acid amplification b. Amplification of the polynucleotides using a polymerase c. Fragmentation of amplified polynucleotides, thereby obtaining a mixture of fragmented and un-fragmented polynucleotides comprising said modified primer d. Ligation of a plurality of adapter oligonucleotides to the mixture obtained in step c), thereby obtaining a mixture of polynucleotides comprising said adapter oligonucleotides, wherein said adapter oligonucleotides comprise a binding site for an amplification primer e. Providing an amplification primer to the mixture obtained in step d), wherein said amplification primer is a starting point for a polymerase for nucleic acid amplification f. initiate a nucleic acid amplification by providing a polymerase Characterized in that the modified primer provided in step a) comprises a functional group # wherein said functional group is a blocking group at the 5′ end of said modified primer, thereby preventing the ligation of the adapter oligonucleotides or # wherein said functional group is at least one nucleotide analogue, and wherein the nucleotide analogue is excised after step d) by an endonuclease, thereby removing the primer binding site provided by the adapter oligonucleotides, thereby preventing binding of the amplification primer provided in step f) and a nucleic acid amplification of fragmented polynucleotides comprising the modified primer