38 results about "Upstream and downstream (DNA)" patented technology

The present application relates to the technical field of bioinformatics, and particularly relates to a method for constructing a biological age prediction model based on DNA methylation. The method comprises the following steps: obtaining whole genome methylation sequencing data of a human peripheral blood sample; classifying cell subpopulations of the human peripheral blood sample, and performing single-cell RNA sequencing processing on each cell subpopulation to obtain single-cell sequencing data including lymphocytes, neutrophils and monocytes; performing tissue-specific analysis on CpG sites within a range of 2000 base pairs upstream and downstream of each cell subpopulation-specific transcription factor binding site according to the single-cell sequencing data and the whole genome methylation sequencing data to obtain candidate marker site data. The present application can provide strong support for early detection of accelerated aging, prediction of related disease risks and guidance of precision medicine.

A sequence for forming an editing substrate together with a target RNA editing site. The sequence and the upstream and downstream portions of the target RNA editing site form a specific double-chain secondary structure, so that efficient editing of the target RNA editing site is realized.

The invention discloses a method for performing gene editing in amycolatopsis orientalis. The method comprises the following steps: (1) constructing upstream and downstream homologous arm fragments nrps8-arm-A and nrps8-arm-Z of a target gene nrps8 sequence for DNA repair after knockout; (2) constructing a DNA (Deoxyribose Nucleic Acid) recombinant fragment gDNA-nrps8 capable of coding the sgRNA; (3) constructing a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 editing plasmid pLYCS01; and (4) electrically transforming the pLYCS01 and the gDNA (deoxyribonucleic acid)-nrps8 into the A.keratiniphila cell, so as to obtain the A.keratiniphila cell. The invention also discloses an application of the nrps8 gene of the A. keratiniphila as a gene knockout target of CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR associated protein 9) in the preparation of vancomycin.

The application discloses a method for simulating high-depth sequencing TSS characteristics. The method comprises the following steps: (1) obtaining reference genome captured free DNA sample sequencing data and sequence coverage of the upstream and downstream regions of the TSS of each gene; (2) obtaining features for constructing TSS values of simulated high-depth sequencing results based on the low-depth sequencing results of free DNA; and (3) constructing a TSS value model of simulated high-depth sequencing results based on the features of the low-depth sequencing results of free DNA. According to the application, the features are extracted based on the low-depth sequencing results, a model is constructed, the TSS characteristics equivalent to high-depth sequencing can be obtained, the number of analyzable gene TSS regions can be effectively increased, the stability of the sequencing fragment coverage of the analyzable gene TSS regions is increased, the sequencing cost is greatly reduced while the calculation accuracy of the TSS values is ensured.

The invention relates to the technical field of bioengineering, and discloses a saccharomyces cerevisiae self-replication system and application thereof.The system comprises a recombinant plasmid, and the recombinant plasmid comprises upstream and downstream homologous arms of a saccharomyces cerevisiae delta site, a selection marker TRP1, an autonomous replication sequence ARS310, a G418 resistance gene and a resistance gene promoter; wherein the nucleotide sequence of the autonomous replication sequence ARS310 is as shown in SEQ ID NO. 3; the nucleotide sequence of the G418 resistance gene is as shown in SEQ ID NO. 4; the nucleotide sequence of the selection marker TRP1 is as shown in SEQ ID NO. 5. The invention provides a pressure-driven saccharomyces cerevisiae self-replicating system for high-efficiency expression of foreign protein (target gene), and the expression of the foreign target gene with high conversion efficiency, high stability and high copy number is realized by utilizing natural multiple copies of a delta site, the self-replicating capability of ARS and the screening pressure of a G418 resistance gene.

The invention provides a method for improving the recombination efficiency of denitrifying hyphomicrobe and application, and belongs to the technical field of gene engineering. On the basis of a pK18mobsacB-Tc vector, a vector capable of simultaneously expressing exonuclease-recombinase and upstream and downstream homologous arms of a target gene is constructed, the vector is transformed into denitrifying hyphomicrobe for homologous recombination, mediation of other plasmids is not needed, traceless knockout of the target gene can be completed only through one round of transformation, and the method is simple and convenient to operate. Compared with existing gene knockout plasmids and transformation methods of denitrifying hyphomicrobe, the method is more efficient. Experiments prove that when the vector is used for constructing a denitrifying hyphomicrobium engineering strain, compared with plasmids without recombinase, the number of recombinants is remarkably increased, and the vector has the characteristics of simplicity, convenience and high efficiency.

The present application relates to a primer set for detecting multiple mutations of human embryo beta-thalassemia, a kit and application thereof. The primer set comprises five primers for amplifying HBB a plurality of mutations in the gene region: HBB-F1, HBB-F2, HBB-F3, HBB-R1 and HBB-R2, the sequences of which are shown in SEQ ID NO: 1-5 respectively; and eight primers for amplifying HBB linkage SNP sites within 1Mb range of the upstream and downstream genes: L1-F, L1-R, L2-F, L2-R, L3-F, L3-R, L4-F and L4-R, the sequences of which are shown in SEQ ID NO: 6-13 respectively. Based on the specific combination of long fragment PCR amplification and long fragment high-throughput sequencing, the present application can simultaneously detect multiple mutations of beta-thalassemia in multiple embryo samples with high specificity, accuracy and speed.

The invention discloses a method for detecting a multi-target gene editing plant based on a liquid-phase gene chip. The invention provides a liquid phase chip. The liquid phase chip comprises a capture probe, the capture probe is designed and synthesized according to the following method: a target region is selected within the upstream and downstream range of not more than 300bp at the position of a to-be-detected site on a reference genome, the target region covers the to-be-detected site, and the capture probe is designed for the target region; the number of the to-be-detected sites is one or more; the capture probe specifically binds to the target region. The capture probe disclosed by the invention is high in variation detection sensitivity, generally high in sequencing depth and reliable in result; variation at different positions of different chromosomes can be detected at the same time, and parallel detection is achieved. According to the detection method based on the liquid-phase gene chip, multi-site, multi-sample and cross-species parallel detection can be realized, and single base mutation can be accurately identified.

The present application relates to the field of DNA methylation modification detection, and particularly relates to nucleic acid sequence molecules and DNA methylation detection methods. The nucleic acid sequence molecules comprise at least a probe sequence, the probe sequence comprises at least one CpG site, at least one of which is a to-be-detected CpG site; the probe sequence comprises a methylation probe sequence and a non-methylation probe sequence, and the two probe sequences are modified with different fluorescent groups; and the probe sequence at least meets one of the following conditions: i) within the region of the to-be-detected CpG site and the 2 nt upstream and downstream of the to-be-detected CpG site, the overall GC content of the methylation probe is not more than 50%; ii) within the region of the to-be-detected CpG site and the 2 nt upstream and downstream of the to-be-detected CpG site, the overall GC content of the methylation probe is more than 50%, and the to-be-detected CpG site in the probe sequence is modified with LNA. The method of the present application can significantly improve the specificity and sensitivity of CpG site methylation detection.

This invention discloses a method based on SOD The primers and methods for gene screening of heat-resistant scallop varieties were developed using scallop cDNA and DNA as templates. PCR amplification was performed using upstream and downstream primers (SEQ ID No. 3 and SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6) to obtain samples containing scallop cDNA and DNA. SOD Gene sequence fragments from the exon and promoter regions were used as molecular markers. The genotypes were CT at the c.229 C>T site, CA at the c.284 C>A site, and CA at the c.348 C>A site in the exon region, and CA at the g.1124 A>C site and AT at the g.1359 T>A site in the promoter region. This method screened for high-temperature resistant scallop varieties, demonstrating advantages such as genetic stability, accuracy, and efficiency.

This article discloses a method for identifying the nature of thyroid nodules, including detecting the DNA methylation level in a sample selected from the following regions (1) and (2): (1) fragments of one or more of the following genes: COL23A1, ILDR2, DHRS3, KIF1A, GDNF, TBX18, and (2) nucleic acid regions within 10 kb upstream and downstream of the genes (1).

The invention belongs to the technical field of gene sequencing, and particularly relates to a populus hybrid parent source identification method, system, equipment and medium based on a single-copy gene sequence, the method comprises the following steps: firstly, constructing a single-copy gene sequence reference library containing five populus reference species, and each sequence comprises a gene coding region and upstream and downstream extension sequences thereof; secondly, acquiring second-generation sequencing data of a to-be-detected sample, comparing and assembling by taking the representative species sequence as reference, and reconstructing a single-copy gene sequence of the to-be-detected sample; and finally, determining a parent source through sequence alignment and statistical analysis. According to the method disclosed by the invention, a single-copy gene sequence can be accurately reconstructed from a hybrid sample through an optimally designed sequence assembly and comparison process, so that the coverage degree and the resolution ratio of a genetic marker are remarkably improved; in addition, a comparison screening rule and a layering judgment standard based on a complete matching length are designed, and precise identification of hybrid parent sources is realized by quantitatively analyzing the matching proportion of each reference species.

This invention provides a primer set, library construction method, and application for whole-genome detection of porcine reproductive and respiratory syndrome virus (PRRSV). The primer set comprises 16 primer pairs, and the sequences of the upstream and downstream primers for each primer pair are shown in SEQ ID NO. 1 to SEQ ID NO. 32. The primer set of this invention exhibits good specificity and sensitivity, and shows promising application prospects in the amplification of the entire PRSV genome and the preparation of reagents or kits required for amplification. The library construction method of this invention includes multiplex PCR amplification using the primer set described herein. This method is compatible with next-generation sequencing platforms and nanopore sequencing platforms, offering a high degree of automation. Even in samples with viral concentrations as low as 1 copy / μL, it can obtain more than 95% of the PRSV sequence, with a sequencing depth exceeding 1000X.

This invention belongs to the field of molecular biology technology, specifically relating to a kit, method, and application for detecting SNP sites. The kit includes: primers, including upstream and downstream primers for amplifying upstream and downstream sequence fragments of the SNP site; probes, including multiple probes that specifically recognize SNP site polymorphisms; multiple different first detection markers, each used to bind to one of the probes; and a second detection marker for binding to the upstream and downstream sequence fragments. By using patterned first detection markers to bind probes for differentiation, a large number of different probes can be mixed in the same reaction well. After the probe captures an amplified fragment containing a specific SNP site, it forms a first detection marker-amplified fragment-second detection marker complex with the second detection marker bound to the amplified fragment. Thus, the genotype of the SNP site can be distinguished based on the combination of the first and second detection markers, ultimately achieving simultaneous and efficient multiplex detection.

This article discloses a method for identifying the nature of thyroid nodules, including detecting the DNA methylation level in a sample selected from the following regions (1) and (2): (1) fragments of one or more of the following genes: GAS6, SOX17, ZMIZ1, TSHR, CDH1, MCRIP2, LINC01977, EGR3, and (2) nucleic acid regions within 10 kb upstream and downstream of the genes (1).

The invention belongs to the technical field of RNA stability detection, and particularly relates to a method for detecting tRNA structure stability based on ribozyme. The invention discovers that when a non-modified site of the tRNA is taken as a cutting target, the tRNA with a more compact space structure can hinder pairing of VMC10A20p to the tRNA and hinder cutting of the VMC10A20p to the tRNA target. By comparing cleavage product levels of tRNA targets of different biological samples, the relative stability of the space structure of the tRNA target is detected. Therefore, the invention provides a method for detecting the tRNA structure, upstream and downstream arms of VMC10A20p are complementarily paired with upstream and downstream of a non-modified site of to-be-detected tRNA, a cleavage sequence is designed for the to-be-detected tRNA site, the to-be-detected tRNA is cleaved, and the structural stability of the tRNA is judged based on a cleavage result.

This application relates to a sequence that forms an RNA editing target site and an editing substrate, belonging to the biopharmaceutical field. The sequence forms a specific double-stranded secondary structure upstream and downstream of the RNA editing target site, thereby enabling efficient editing of the RNA editing target site.

The invention provides a sequencing method and application thereof. The sequencing method comprises the following steps: 1) establishing a library for long fragment DNA to obtain a sequencing library; 2) loading and sequencing the sequencing library, keeping a proximity relationship between clusters generated by library molecules derived from the same long fragment DNA in the flowcell, and obtaining a read sequence and a position coordinate of the read sequence on the flowcell; wherein the sequencing library retains upstream and downstream associated information of DNA in library molecules in the long fragment DNA. According to the sequencing method, the upstream and downstream associated information in the sequencing library is converted into the distance of the cluster position, and the linkage relationship between reads is restored through information analysis, so that higher genome loading capacity can be realized.

The present application relates to a primer set for detecting multiple mutations of human embryo beta-thalassemia, a kit and application thereof. The primer set comprises five primers for amplifying HBB a plurality of mutations in the gene region: HBB-F1, HBB-F2, HBB-F3, HBB-R1 and HBB-R2, the sequences of which are shown in SEQ ID NO: 1-5 respectively; and eight primers for amplifying HBB linkage SNP sites within 1Mb range of the upstream and downstream genes: L1-F, L1-R, L2-F, L2-R, L3-F, L3-R, L4-F and L4-R, the sequences of which are shown in SEQ ID NO: 6-13 respectively. Based on the specific combination of long fragment PCR amplification and long fragment high-throughput sequencing, the present application can simultaneously detect multiple mutations of beta-thalassemia in multiple embryo samples with high specificity, accuracy and speed.

The application discloses a method for constructing a directional SNP mutant in Neurospora crassa, and the method comprises the following steps: designing and synthesizing a forward primer containing a target SNP and a reverse primer located downstream of a 3'-UTR of a gene, and amplifying a gene segment A containing a mutation site from genomic DNA as a template; respectively amplifying homologous arm segments B and C located upstream and downstream of the segment A, wherein a reverse primer of the segment B comprises a reverse complementary sequence of a forward primer of the segment A; amplifying a hygromycin B resistance gene from a plasmid pCSN44 as a template hph , wherein primers at two ends of the hygromycin B resistance gene are respectively reverse complementary to the reverse primer of the segment A and the forward primer of the segment C; fusing the segments B, A, hph and C into a fusion segment B-A-hph-C through fusion PCR; transforming the fusion segment into the Neurospora crassa through electroporation, and obtaining an SNP site-directed mutant strain through hygromycin resistance screening and first-generation sequencing verification. The application has the advantages of simple operation, high mutation efficiency and accurate site, and provides a reliable technical means for gene function research of the Neurospora crassa.

The invention provides a detection kit for detecting interferon-related activating genes in neutrophil in a tumor microenvironment. The detection kit comprises an RNA extraction reagent, a reverse transcription reaction reagent, a quantitative PCR reagent, a probe, a positive reference substance and a negative reference substance. And the quantitative PCR reagent comprises upstream and downstream primers of nine interferon related genes, namely, ISG15 (Interferon Sorting Gene 15), RSAD2 (Registered Sequence Administration 2), IFIT1 (Interferon Institute of Transcription 1), IFI44L (Interferon Institute 44L), MX1, OAS1, STAT1, IRF7 and CXCL10. The detection kit can be used for detecting the expression quantity of nine genes including ISG15, RSAD2, IFIT1, IFI44L, MX1, OAS1, STAT1, IRF7 and CXCL10, accurate curative effect grouping is realized through dynamic monitoring of ISG15 + neutrophile granulocyte subpopulation, and meanwhile, improvement of curative effect prediction capability can be realized through combination with mrTRG.

The invention discloses a single-gene hereditary cardiovascular disease related gene detection probe group, a kit and an application of the single-gene hereditary cardiovascular disease related gene detection probe group. The probe group is a specific probe which is selected from exon regions of genes such as ABCC9, ABCG8, ACADM, ACADS, ACADVL, ACTA2, ACTC1, ACTN2, ACVRL1, AGK, AGL, AGXT, AIFM1 or ALMS1 and the like, and 20-30 bp intron regions on the upstream and downstream of the exon regions of the genes such as ABCC9, ABCG8, ACADM, ACADDS, ACADVL, ACTA2, ACTC1, ACTN2, ACVRL1, AGK, AGL, AGXT, AIFM1 or ALMS1. The detection probe and the kit provided by the invention can accurately detect exon regions of 268 pathogenic gene variations related to the single-gene hereditary cardiovascular disease, intron regions of 20-30 bp upstream and downstream of exon and copy number variations of pathogenic genes, under the average 1G data volume, the nuclear genome coverage depth reaches 200 * on average, the 30 * coverage degree reaches 99%, and the detection sensitivity is high. The detection sensitivity of low-frequency mutation in a target area is improved, the detection cost is effectively controlled, an experimental system is stable, and the discrete coefficient of library results among samples is less than 10%.

The invention relates to the technical field of gene engineering, and discloses a protein salt purification vector construction method and application of a constructed vector in a kit, the construction method comprises the following steps: designing primers according to upstream and downstream sequences of a target gene to be inserted; carrying out annealing treatment on the primer to form a DNA template to be inserted; the method comprises the following steps: mixing a double-stranded DNA template, a plasmid vector pET28a-icSAT, restriction enzyme, heat-resistant T4DNA ligase, a target gene and a buffer solution to construct a vector; and transforming the vector into a competent cell E.coli DH5alpha, and amplifying to obtain a protein salt purified vector capable of being inserted into a target DNA sequence. By adopting the construction method, the insertion of the target gene and the construction of the vector are synchronously completed in a single reaction system, the steps of multiple rounds of enzyme digestion, connection and gel purification in a traditional method are omitted, and the construction efficiency is remarkably improved; meanwhile, the target gene can be connected into a corresponding expression vector without PCR amplification.

The invention discloses an accurate sequence analysis method and system based on nanopore length and read length sequencing data. The method comprises the following steps: comparing nanopore sequencing data with a reference genome through a minimap2 algorithm, and screening out high-quality long-read-length sequences in a target area and specific upstream and downstream ranges; extracting a target area local sequence of each read length, calculating an editing distance matrix, performing Box-Cox transformation, performing clustering analysis by adopting a Gaussian mixture model, and determining an optimal clustering number through a BIC criterion; performing multi-sequence alignment and polishing on each type of sequences to generate a high-precision consensus sequence; and finally, recognizing homologous conserved sequence fragments through re-comparison and global comparison, and outputting candidate sequences for targeting PCR (Polymerase Chain Reaction) primer or CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) sgRNA design. According to the method, the problem of high error rate of nanopore length and read length data can be effectively solved, the accuracy and reliability of sequence analysis are improved, and reliable technical support is provided for precise gene editing and molecular diagnosis.

The invention relates to the technical field of DNA cloning, and discloses a universal short-fragment DNA cloning method based on reverse PCR and template-free PCR and application of the universal short-fragment DNA cloning method, and the universal short-fragment DNA cloning method comprises the following cloning steps: taking an annular plasmid as a template, and adopting reverse PCR amplification to obtain a linearized vector; an upstream primer and a downstream primer are designed and synthesized, the 3'ends of the two primers have complementary overlapping regions, the 5 'ends of the two primers contain plasmid homologous sequences, and the two primers are subjected to thermal cycle self-assembly and extension in a PCR reaction system without exogenous template DNA to form a blunt-end double-stranded DNA fragment; and purifying the blunt-end double-stranded DNA fragment and a linearized vector, connecting through homologous recombination, and transforming a host cell to obtain a recombinant plasmid containing a target gene. The universal cloning method for the short-fragment DNA does not depend on any restriction enzyme cutting site, and the vector is universal; meanwhile, an exogenous template DNA is not needed; time and cost are greatly reduced, and the method is suitable for construction of high-throughput sgRNA, epitope tags, promoter elements and the like.

The invention discloses a self-assembly self-replication RNA (Ribonucleic Acid) system and application thereof. Belongs to the technical field of biological medicine. The method comprises the following steps: cutting off self-replicating RNA into two independent RNA sequences, and respectively accessing RNA ribozyme sequences to the upstream and downstream of each cut-off part; after the independent RNA is transcribed in vitro, ribozyme is autocatalytically broken to generate 2 ', 3'-cyclic phosphoric acid and a 5 '-hydroxyl terminal, the 2', 3 '-cyclic phosphoric acid and the 5'-hydroxyl terminal are introduced into a cell and are self-linked through host RTCB ligase to form complete saRNA, and efficient and continuous expression of target protein is realized. According to the present invention, through the splitting-self-assembly modular design, the length of the single RNA is reduced to 3000-7600 nt, the in vitro transcription efficiency is improved by 3-10 times, and the encapsulation of the lipid nanoparticles is easily achieved. The bottleneck that the traditional saRNA is large in molecular weight, difficult to produce and low in delivery efficiency is solved, and the method has important application value in the fields of vaccine production, recombinant protein expression, cell reprogramming and gene therapy.

The application discloses an isothermal amplification and detection technology based on CRISPR-chain substitution. The isothermal amplification kit based on CRISPR-chain substitution comprises chain substitution isothermal amplification reagents, and the kit further comprises wild-type Cas9 and / or nuclease-deficient Cas9 nickase, an sgRNA pair for specifically recognizing the upstream and downstream of a target DNA sequence, and an amplification primer pair for chain substitution isothermal amplification reaction, wherein the primer pair is complementary to a single-stranded region exposed after the Cas9-sgRNA complex is combined with the target gene. The technology is simple in operation, strong in anti-interference capability, and accurate and reliable in detection result.

The application discloses a crop mitochondrial editing site prediction method and system and a storage medium. Mitochondrial genome sequences of multiple species and corresponding C-to-U RNA editing site annotation information are obtained. Fragments of a set length are taken from the upstream and downstream of the mitochondrial genome sequence and the corresponding C-to-U RNA editing site annotation information sequence as positive samples with the editing site as the center. Any site without editing site annotation is taken as the center, and fragments of a set length are taken from the upstream and downstream of the mitochondrial genome sequence and the corresponding C-to-U RNA editing site annotation information as negative samples. Part of the positive samples and the negative samples corresponding to each species are used to construct a training set of each species, the training set of each species is used as the input of a convolutional neural network, and the optimal prediction model corresponding to each species is obtained. The application has high prediction accuracy and can better fit the potential encoding mechanism of C-to-U RNA editing of different species.

The invention relates to the technical field of molecular biology, and discloses a PCR chip and a kit for detecting the expression quantity of functional genes related to biodegradation of brominated compounds and application of the PCR chip and the kit, and the PCR chip comprises primer pairs for respectively and specifically amplifying genes PbrA1, PbrA2, PbrA3 and RHDA; the nucleotide sequences of an upstream primer and a downstream primer of the primer pair for specifically amplifying the PbrA1 gene are as shown in SEQ ID NO.1-2; the nucleotide sequences of an upstream primer and a downstream primer of the primer pair for specifically amplifying the PbrA2 gene are as shown in SEQ ID NO.3-4; the nucleotide sequences of an upstream primer and a downstream primer of the primer pair for specifically amplifying the PbrA3 gene are as shown in SEQ ID NO.5-6; the nucleotide sequences of an upstream primer and a downstream primer of the primer pair for specifically amplifying the RHDA gene are as shown in SEQ ID NO.7-8. The PCR chip can be used for accurately detecting the expression condition of functional genes related to the reductive dehalogenation effect of the brominated compound, so that the PCR chip can be used for reflecting the pollution and biodegradation conditions of the brominated compound and exploring the bioremediation feasibility of the pollution of the brominated compound.

A sequence that forms an editing substrate with an RNA editing target site. The sequence and upstream and downstream of an RNA editing target site form a specific double-chain secondary structure, so that efficient white-spoon editing of the RNA editing target site is realized.