55 results about "Primer binding site" patented technology

The present disclosure provides methods for conducting in situ multiplex and multi-omics detection and identification using coded padlocks probes. The methods comprise simultaneous use of RNA-specific padlock probes and polypeptide-specific padlock probes to detect both RNA and polypeptides in a cellular sample. Both types of probes include a barcode that unique identifies the RNA or polypeptide that that padlock probe detects. Both types of probes also include a batch-specific sequencing primer binding site to enable sequencing a desired subset of concatemer template molecules. Use of the batch-specific sequencing primers reduces overcrowding signals and images, to produces optical images that are intense and resolvable. By conducting multiple rounds of sequencing on the same cellular sample using different batch-specific sequencing primers enables multiplex and multi-omics sequencing to reveal numerous target RNAs and their encoded polypeptides.

The invention discloses pegRNA of a targeted TRPV4 gene, a pilot editing system and application of the pegRNA and the pilot editing system. Specifically, the invention discloses pegRNA which comprises sgRNA, an RT template and a primer binding site, and the nucleotide sequence of the RT template is 108th to 121st sites of SEQ ID No.2. And the nucleotide sequence of the pegRNA is SEQ ID No. 2. The invention further discloses a pilot editing system and an editing method for repairing the TRPV4 gene V342F mutation. The screened specific pegRNA and a pilot editing system can efficiently realize correction of the TRPV4 gene, the purpose of repairing V342F mutation of the TRPV4 gene of the Scottian folding cat is achieved, fewer by-products are obtained, the highest editing efficiency can reach 41%, and the Scottian folding cat has important clinical value and application prospects in treatment of osteochondral dysplasia of the Scottian folding cat.

Provided herein is a nucleic acid detection system comprising: a CRISPR system comprising an effector protein and one or more guide RNAs designed to bind to corresponding target molecules; an RNA-based masking construct; and optionally, nucleic acid amplification reagents to amplify target RNA molecules in a sample. In another aspect, the embodiments provide a polypeptide detection system comprising: a CRISPR system comprising an effector protein and one or more guide RNAs designed to bind a trigger RNA, an RNA-based masking construct; and one or more detection aptamers comprising a masked RNA polymerase promoter binding site or a masked primer binding site. In some embodiments, the system may be used to detect viruses in samples.

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.

The present disclosure provides methods for in situ multiplex and multi-omics detection and identification using coded padlock probes. The method includes detecting both RNA and polypeptide in a cell sample using both an RNA-specific padlock probe and a polypeptide-specific padlock probe. The two types of probes include barcodes that uniquely identify the RNA or polypeptide detected by the padlock probe. The two types of probes also include batch-specific sequencing primer binding sites to enable sequencing of a subset of desired concatemer template molecules. The use of batch-specific sequencing primers reduces over-crowded signals and images to produce strong and distinguishable optical images. Multiple rounds of sequencing are performed on the same cell sample by using different batches of specific sequencing primers, so that multiple and multi-omics sequencing can reveal numerous target RNAs and polypeptides encoded by the target RNAs.

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, wherein 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 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.

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 are modular prime editing systems, comprising: i) a fusion protein comprising a Cas9 nickase protein linked to a nucleotide polymerase (NT) protein, ii) a prime editor template RNA (petRNA) comprising a primer binding site (PBS), a nucleotide polymerase template (NPT), and at least one MS2 hairpin, and iii) a single guide RNA (sgRNA).

The invention provides methods of generating forward and reverse reads of an immobilized single-stranded target nucleic acid using multifunctional primers. A multifunctional primer serves to initiate separate syntheses of first and second complementary strands to the single-stranded target nucleic acid, and to tether the first complementary strand to the immobilized target nucleic acid. The first complementary strand serves to provide a primer binding site and template for an extension product to generate a reverse sequencing read. The second complementary strand serves to displace the first complementary strand from duplexing with the target nucleic acid. The first or second complementary strand can provide a forward sequence read.

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.

Provided are modular prime editing systems, comprising: i) a fusion protein comprising a Cas9 nickase protein linked to a nucleotide polymerase (NT) protein, ii) a prime editor template RNA (petRNA) comprising a primer binding site (PBS), a nucleotide polymerase template (NPT), and at least one MS2 hairpin, and iii) a single guide RNA (sgRNA).

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 present invention relates to a method for obtaining a nucleic acid library of a sample comprising a polynucleotide, the method comprising the steps of: a. Providing the polynucleotide with a plurality of modified primers wherein the modified primers are the starting point of a polymerase for nucleic acid amplification; b. Amplifying the polynucleotide using a polymerase; c. Fragmenting the amplified polynucleotide, thereby obtaining a mixture of fragmented and unfragmented polynucleotides comprising the modified primer; d. Linking a plurality of linker oligonucleotides to the mixture obtained in step c), thereby obtaining a mixture of polynucleotides comprising said linker oligonucleotides, said linker oligonucleotides comprising binding sites for amplification primers; e. Providing an amplification primer to the mixture obtained in step d), wherein the amplification primer is the starting point of the polymerase for nucleic acid amplification; f, nucleic acid amplification is started by providing polymerase; characterised in that the modified primer provided in step a) comprises a functional group wherein the functional group is a blocking group located at the 5'end of the modified primer, thereby preventing the ligation of linker oligonucleotides, or wherein the functional group is at least one nucleotide analogue, thereby preventing the ligation of linker oligonucleotides. And wherein the nucleotide analogue is resected after step d) by an endonuclease, thereby removing the primer binding site provided by the linker oligonucleotide, thereby preventing binding of the amplification primer provided in step f) and nucleic acid amplification of the fragmented polynucleotide comprising the modified primer.

The present invention relates to the field of ligation of oligonucleotides to DNA fragments. The ligation method of the present invention can be used, for example, to attach an oligonucleotide containing an adapter, primer binding site, promoter, tag, barcode, or any combination of the above to a DNA fragment.

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.

The invention provides methods of generating forward and reverse reads of an immobilized single-stranded target nucleic acid using multifunctional primers. A multifunctional primer serves to initiate separate syntheses of first and second complementary strands to the single-stranded target nucleic acid, and to tether the first complementary strand to the immobilized target nucleic acid. The first complementary strand serves to provide a primer binding site and template for an extension product to generate a reverse sequencing read. The second complementary strand serves to displace the first complementary strand from duplexing with the target nucleic acid. The first or second complementary strand can provide a forward sequence read.

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).

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.

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.

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.

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.