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

The invention belongs to the field of application of microbial genetic engineering and metabolic engineering, and particularly relates to a method for improving the biosynthesis efficiency of sclareol. Specifically, in a host strain, gene knockout is realized through a CRISPR / Cas9 technology, or / and a DNA fragment is introduced, and a strain metabolic pathway is adjusted to obtain a corresponding engineering strain; carrying out biosynthesis by utilizing the obtained engineering strain to obtain sclareol; the gene knockout is realized by introducing donor DNA (Deoxyribose Nucleic Acid) containing upstream and downstream homologous arms of a coding frame; the DNA fragment sequentially comprises an upstream homologous arm, a promoter, a gene, a terminator and a downstream homologous arm from upstream to downstream, the host strain is selected from any one of saccharomyces cerevisiae. According to the method, the cell life is associated with product synthesis, and meanwhile, metabolic balance is coupled, so that the synthesis efficiency of a target product is improved, and a foundation is laid for industrial fed-batch fermentation.

The invention relates to a framework based on a miR-30 framework, a construction method of the framework and application of the framework in RNAi (Ribonucleic Acid Interference). The skeleton construction method comprises the following steps: taking an existing shRNAmiR30 vector as a PCR (Polymerase Chain Reaction) template; the method comprises the following steps: designing two pairs of complementary overlapping primers of siRNA containing a target gene, namely primers F1 and R1 and primers F2 and R2, respectively performing amplification, and performing OE-PCR by taking the obtained P1 and P2 as templates and the primers F1 and R2 as upstream and downstream primers to obtain miR-OE. Furthermore, according to the construction method disclosed by the invention, reverse mutation of a stem-loop structure in a miR-OE skeleton and an EcoRI restriction enzyme cutting site is also carried out. The primers required to be synthesized in the miR-OE skeleton and miR-OE-EL construction method are short chains with the length of less than 60nt, the synthesis difficulty and cost are effectively reduced, and the primer design is more flexible; moreover, the interference efficiency of the miR-OE-EL skeleton on the target gene is obviously improved, and the miR-OE-EL skeleton can be well applied to preparation of shRNA drugs.

The invention discloses a recombinant III-type human-like collagen as well as a coding gene and an expression method thereof, and the method comprises the following steps: S1, carrying out sequence optimization by taking a human III-type collagen sequence as a template to obtain a recombinant III-type human-like collagen gene sequence; designing PCR (Polymerase Chain Reaction) amplification upstream and downstream primers according to the gene sequence, and amplifying the gene sequence of the recombinant III type human-like collagen; s2, carrying out double enzyme digestion on the recombinant III type human-like collagen gene and a plasmid, and connecting to obtain a recombinant plasmid; s3, linearizing the recombinant plasmid, transforming the linearized recombinant plasmid into agrobacterium, and screening; and S4, infecting lettuce with agrobacterium, culturing to express protein, freezing the infected lettuce with liquid nitrogen, grinding, extracting total plant protein, and purifying to obtain the recombinant III-type human-like collagen. The recombinant III type human-like collagen is expressed through lettuce, so that the method is safe and efficient, and has sufficient market value and important practical significance.

The invention belongs to but not limited to the technical field of genetic engineering technology, and discloses a construction method and application of a BspE gene deletion strain of Brucella effect protein, the BspE gene deletion strain is constructed by using homologous recombination and SacB reverse screening technology, Brucella abortus A19 vaccine strain liquid is used as a template, PCR amplification is carried out to obtain upstream and downstream homologous arms of BspE gene, and the BspE gene deletion strain is constructed by using the upstream and downstream homologous arms of the BspE gene. The method comprises the following steps: carrying out fusion PCR (Polymerase Chain Reaction) amplification on upstream and downstream homologous arms of a BspE gene subjected to gel recovery, and connecting and transforming an obtained fusion fragment and a pUC19-SacB plasmid to obtain a recombinant plasmid pUC19-delta BspE. The invention relates to a bovine brucella BspE deletion vaccine strain, which is characterized in that a plasmid is obtained, the plasmid is electrically transformed into a bovine brucella A19 competent cell, and the positive transformant is a bovine brucella BspE deletion strain through kanamycin resistance and sucrose pressure screening, PCR (polymerase chain reaction) identification and sequencing, and the deletion vaccine strain improves the levels of antibacterial cytokines and specific brucella antibody IgG, and can be used for preparing the bovine brucella BspE deletion vaccine strain. The Brucella attenuated vaccine strain is a novel Brucella candidate attenuated vaccine strain and has a good market prospect.

The invention discloses a gene editing method of lactic acid bacteria from activated sludge, and belongs to the technical field of gene engineering. The invention provides a lactobacillus plantarum gene editing system based on original bacteriophage recombinase combined with a cre-lox system, which is characterized in that a fusion linear double-stranded DNA (deoxyribonucleic acid) fragment containing upstream and downstream homologous arms of a target gene and a resistance gene with a lox site is transformed into lactobacillus plantarum for expressing the original bacteriophage recombinase; and genome editing is realized by a homologous recombination method. In addition, the resistance screening marker is recovered by inducing transient expression of Cre enzyme, so that subsequent continuous editing of other genes is facilitated.

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.

The invention relates to the field of genetic engineering, in particular to application of a tandem expression module based on ribosome recovery factor antisense RNA, and the tandem expression module is used for biosynthesis of a regulatory element in cells and regulation of the upstream and downstream protein expression proportion of a tandem site under the condition of protein tandem expression. The combination of the antisense RNA sequence, the expression structure of the antisense RNA sequence and the linker sequence between the expression cassettes can effectively adjust the relative expression proportion of tandem expression upstream and downstream proteins, and is used for gene function circuits with related requirements and optimal expression proportion exploration for specific protein pairs.

The invention discloses a primer, a kit and a method for identifying Cobitidae fish. The identification primers include a first primer pair and a second primer pair, the upstream and downstream of the first primer pair respectively have nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2, and the upstream and downstream of the second primer pair respectively have nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4. The method for identifying Cobitidae fish includes: S1: obtaining DNA of a Cobitidae fish sample; S2: performing PCR amplification on the DNA obtained in S1 with the first primer pair to obtain a first PCR amplification product; S3: performing nested PCR amplification with the second primer pair, using the first PCR amplification product as a template to obtain a second PCR amplification product; S4: performing agarose gel electrophoresis on the second PCR amplification product obtained in S3, cutting and purifying the gel, and obtaining a purified second PCR amplification product; and S5: sequencing the purified second PCR amplification product, comparing the mitochondrial gene sequence of the known species of Cobitidae fish in the NCBI database according to the base sequence of the sequencing result, and confirming the species of the Cobitidae fish sample.

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 belongs to the technical field of animal genome editing, and particularly relates to a method for realizing fixed-point integration of full-length exogenous DNA fragments by using long single-stranded DNA. The technical problem to be solved by the invention is to provide a method for efficiently realizing site-specific integration of an exogenous full-length gene in a mammal genome. According to the technical scheme, the method for realizing fixed-point integration of the full-length exogenous DNA fragment by using the long single-stranded DNA comprises the following steps: converting a mixture of sgRNA, Cas9mRNA and / or Cas9 protein of a long single-stranded exogenous gene and guiding Cas9 to cut an integration site into recipient cells; the exogenous gene long single strand comprises a 5'homologous arm and a 3 'homologous arm which are arranged on the upstream and downstream of an sgRNA guide cleavage site, a full-length exogenous DNA fragment sequence arranged between the 5' homologous arm and the 3 'homologous arm, and related matching original parts required by expression of the full-length exogenous DNA fragment sequence.

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 present invention relates to a DNA probe, a kit and a method for distinguishing between fruit-type and vegetable-type papaya varieties, wherein the nucleotide sequence of the DNA probe is shown in SEQ.ID.NO.1. Based on the alignment results of the chloroplast genome sequences of various papaya varieties, we found a plurality of differential sites, among which there is a differential site in the region near 60‑70 b. Through this one site, we can distinguish papaya varieties with different production uses, which is an ideal site for molecular markers. We intercepted a sequence of about 150 bp upstream and downstream of the differential site as a reference sequence, selected homologous sequences to design primers, and used the DNA of samples of various papaya varieties as templates for PCR amplification. A single bright band consistent with the expected size can be amplified in all 7 templates, proving that the differential site fragment obtained by analyzing the chloroplast genome sequence exists and can be amplified and identified, and an oligonucleotide fluorescent probe is designed based on the site.

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 invention discloses an in-situ capture, amplification and sequencing method of an RNA (Ribonucleic Acid) target sequence, which is characterized in that a lock-type probe with a gap is designed aiming at the RNA target sequence, the 5'end and the 3 'end of the lock-type probe are respectively complementarily hybridized with the upstream and downstream of the target sequence, and a middle gap corresponds to the target sequence. CDNA of a target sequence is generated through reverse transcription, and the cDNA is connected with the 5'end of the probe through DNA ligase to form annular DNA. And after rolling circle amplification, target sequence information is obtained through in-situ sequencing. According to the method, the experimental process is simplified, the detection sensitivity and specificity are improved, the method is suitable for various sample types, and an efficient tool is provided for spatial transcriptomics research.

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 improving precise traceless gene editing efficiency of yarrowia lipolytica, and belongs to the field of microbial gene engineering. The method for improving the precise traceless gene editing efficiency of the yarrowia lipolytica comprises the following steps: constructing a free gene editing plasmid containing recombinase and upstream and downstream homologous sequences of a sequence to be edited, and culturing after plasmid transformation. The method does not need to inhibit an NHEJ path, the finally obtained engineering bacteria do not leave redundant sequences or plasmids, and the precise gene editing efficiency of the yarrowia lipolytica can be obviously improved only through a single carrier.

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.

Provided is a vector. The vector comprises: i) a replication origin; ii) a first terminator; iii) a) a target DNA sequence, comprising a DNA sequence capable of forming a non-B DNA conformation, or b) a target DNA expression cassette, comprising a promoter and a target DNA sequence, wherein the target DNA sequence comprises a DNA sequence capable of forming a non-B DNA conformation; iv) a second terminator; and v) a selectable marker and a promoter thereof, wherein in a replication direction, the replication origin is at least 150 bp apart from the DNA sequence capable of forming the non-B DNA conformation, the first terminator and the second terminator are respectively located upstream and downstream of the target DNA sequence in a), or the first terminator and the second terminator are respectively located upstream and downstream of the target DNA expression cassette in b). The provided vector can improve the amplification and / or expression (transcription) stability of poly(A) tail and ITR sequences.

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).

A RPA primer probe, a kit and a detection method for detecting two-pointed meiqi yeast belong to the field of biotechnology. The kit is an RPA-LFD kit, which includes a composition of RPA primers and RPA probes. The primers are based on the target gene sequence of two-pointed meiqi yeast of Chinese mitten crab, and the 5' end of the downstream primer is labeled with a modification group-biotin; the upstream and downstream primers with a synthesized amplification fragment size of 292bp. The 5' end of the RPA probe is labeled with carboxyfluorescein FAM, the 3' end is added with an extension blocking group C3Spacer, and a tetrahydrofuran THF is added in the middle of the 5' end and the 3' end. The present invention can realize the RPA detection of two-pointed meiqi yeast, and can quickly, simply and specifically detect two-pointed meiqi yeast.

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.

The invention provides a probe set for detecting the copy number of an NDUFA1 gene and application of the probe set. The probe group comprises No.1, No.2 and No.3 exons of the NDUFA1 gene, No.1 and No.2 introns and probes in 400bp-1000bp of upstream and downstream regions of the gene. The invention shows the advantages of NDUFA1 gene detection in the aspects of specificity, sensitivity, repeatability and convenience, and particularly provides an accurate, reliable and efficient detection method in the aspects of detecting gene copy number variation and solving the problem of a complex gene structure.