39 results about "GC-content" patented technology

The application relates to a sarcoma fusion gene nanopore sequencing primer set and application thereof, wherein the primer set comprises five pairs of specific primers (Primer 1-5) with fixed sequences, each primer follows the design principle of 18-25 bp in length, 40%-60% in GC content and strict matching of a target sequence at a 3' end, and the length of an amplification product is 300-800 bp, which is suitable for nanopore sequencing. Through qPCR verification, the primer set has single-peak melting curves, close-to-100% amplification efficiency, strong specificity and excellent amplification performance.

The application discloses a kind of compositions, reagents and its detection method for high-throughput gene sequencing, belong to gene sequencing technical field, the composition includes Tris-HCl buffer 80-120 parts, potassium chloride 40-60 parts, ammonium sulfate 10-20 parts, magnesium chloride 15-25 parts, dNTPs mixed solution 30-50 parts, heat-stable DNA polymerase mutant 8-15 parts, PCR promoter complex 5-12 parts, modified iridium quinoline ketone complex 1-5 parts, glycerol 150-250 parts, Tween-20 1-3 parts, EDTA disodium salt 0.5-2 parts and nuclease-free ultrapure water, by introducing the modified iridium quinoline ketone complex of innovation, effectively solve the technical problems of low amplification efficiency, poor multiplex PCR reaction uniformity and high non-specific amplification background for high GC content, complex secondary structure genomic template, significantly improve the specificity of amplification, uniformity and the quality and reliability of final sequencing data.

A microbial gene editing scheme automatic design method, system and storage medium; the method comprises: selecting a design object for different types of strains, the design object is bacteria or fungi; after determining the target gene, obtaining the target gene information from the NCBI database; the design scheme of bacteria comprises the following steps: determining the gRNA design region; selecting the optimal gRNA sequence; doing sequence complexity and GC content analysis; the design scheme of fungi uses a frameshift scheme: the gRNA of the frameshift scheme is set at any position not overlapping with other genes after the start codon in the gene; select gRNA sequence in gRNA design region, select the optimal gRNA sequence; design two pairs of four primer sequences for homologous arm identification primer; this scheme can replace the complicated and time-consuming manual scheme, save a lot of time, solve the problem of high cost in the design of the best site and the design of sgRNA sequence scheme of artificial gene knockout.

The application belongs to the technical field of gene detection, and discloses a method and system for judging the influence of homologous regions in NGS gene variation detection. The method comprises the following steps: obtaining a first type of gene and a plurality of second type of genes according to a public database; obtaining corresponding original sequence files of the first type of gene and the second type of gene based on a local gene database, obtaining corresponding sequence alignment files based on a local BAM database, and combining them to obtain known NGS gene data; calculating the GC content, the proportion of repeat units, and the similarity ratio in the original sequence file, and calculating the alignment quality in the corresponding sequence alignment file to construct a training sample; inputting the training sample into a classification model to obtain an optimized classification model; extracting the GC content, the proportion of repeat units, the similarity ratio, and the alignment quality from the NGS gene data to be detected and inputting them into the optimized classification model to judge whether the variation detection is affected. The application can judge the influence of homologous regions in variation detection without the aid of prior information of homologous regions.

The present invention relates to a Klotho messenger-RNA (mRNA), wherein the mRNA has a 5' CAP region, a 5' un-translated region (5'-UTR), a coding region encoding a Klotho polypeptide, a 3' untranslated region (3'-UTR) and a poly-adenosine Tail (poly-A tail), wherein the Klotho polypeptide comprises the KL1 domain of human Klotho, preferably wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having at least 80 % sequence identity to SEQ ID NO: 1, and wherein the coding region encoding the Klotho polypeptide preferably has a GC content of at least 54 %.

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.

The invention belongs to the technical field of gene engineering, and particularly relates to an arthrobacter carbodii gene knockout vector and method. The vector comprises a target gene upstream and downstream homologous arm amplification sequence and a target gene double PAM integration fragment; wherein the target gene is a gene segment to be knocked out; the lengths of upstream and downstream arms of the target gene are respectively 1000bp, and the lengths of amplified homologous arm genes are respectively 2000bp; a basic group at the tail of the sequence of the double PAM integrated fragment is adenine or guanine, and the GC content is 40-60%. According to the knockout vector and the corresponding knockout method, the stable knockout of the gene in the Arthrobacter carbodii can be realized, the knockout efficiency is high, and the knockout vector and the corresponding knockout method have important significance for researching the gene function of the Arthrobacter carbodii and also have a certain reference value for other strains of the Arthrobacter.

The invention belongs to the technical field of biology, and particularly relates to a DNA polymerase mutant for improving amplification efficiency of a high GC sample and application of the DNA polymerase mutant. According to the invention, DNA polymerase mutants KODG and KODC are obtained through site-specific mutagenesis, and on the basis of retaining the capability of amplifying a DNA template with conventional GC content, the capacity of amplifying a DNA template with high GC content (gt; compared with the prior art, the method has the advantages that the amplification efficiency of a DNA template (3kb, 60%) is improved, effective amplification of difficult-to-amplify fragments with 3kb, 95% GC content and the like is successfully realized, and the technical bottleneck of amplification failure caused by high thermal stability and difficulty in complete melting of the existing polymerase in a high-GC region is overcome, so that the application range of the DNA polymerase in complex template amplification is expanded, and the application prospect is broad. And a more reliable enzymology tool is provided for related detection and identification of high-GC-content samples.

The application discloses a wheat salt-tolerant related gene DNA sequence capture sequencing method and belongs to the technical field of molecular biology and genomics. The method comprises the following steps: synthesizing a group of biotin-labeled DNA capture probes according to the salt-tolerant related gene candidate region coordinate interval, the probes are unevenly spaced in the target region, the interval distance is dynamically adjusted by the GC content fluctuation curve in the region, and the probe density is increased when the gradient change rate of the GC content is greater than a threshold value; taking wheat genome DNA as a template, the probes are competitively hybridized with the denatured genome DNA in a liquid phase system to form a target-probe complex; the complex is captured by a streptavidin magnetic bead, non-specific binding fragments are removed through gradient warming and washing, and target DNA is collected; the target DNA is subjected to PCR amplification, purification, construction of a sequencing library and double-end sequencing, unique alignment reads are extracted after alignment, and a genotype state is calculated according to the coverage depth distribution of the target site.

The invention discloses a method and a system for realizing DNA storage by aiming at multi-rule rotation coding of Chinese texts, and relates to the technical field of DNA storage. Encoding the Chinese text by using a five-stroke font input method; mapping is carried out according to the positive and negative code tables, and an interval balance and dynamic detection strategy is introduced in the mapping process to control GC content distribution in intervals and the probability of occurrence of homopolymers between the intervals. Scattering and recombining the sequence by using block coding, and compressing by using RLE coding, wherein the RLE coding generates a sequence file and a run-length file; performing GC content constraint on the sequence file by using cross coding; and br compression is adopted for the run-length file to further improve the compression ratio. And generating a DNA sequence through rotary coding. According to the method, five-stroke coding, positive and negative code table mapping and sequence reconstruction strategies are introduced, so that the information storage density is higher, the local GC content of the DNA sequence is more stable, the homopolymer length is smaller, the unexpected motif proportion is lower, and the data has higher reliability and safety in the storage process.

This invention relates to a kit and method for detecting pathogenic genes of X-chromosome-linked hereditary retinal degenerative diseases. The kit includes a library hybridization reagent and a probe set for detecting XLIRD pathogenic genes, including capture probes that can simultaneously and specifically capture the RPGR, RP2, RS1, and CHM pathogenic genes. The capture probes for capturing the RPGR pathogenic gene include probes with sequences as shown in SEQ ID NO. 1-96, and capture probes targeting the high-GC region of the RPGR gene with sequences as shown in SEQ ID NO. 24-55. This invention targets the above four pathogenic genes, comprehensively analyzes their variation profiles and sequence characteristics, optimizes the amplification region range, and designs a high-density capture probe set including exons, introns, UTR regions, and upstream and downstream 20kb regions. Specifically targeting the high-GC content region of exon 15 of the RPGR gene, the probe sequence and capture strategy are adjusted to achieve efficient capture of the high-GC region. During the library PCR enrichment stage, the amplification enzymes in the system are screened and optimized. Combined with the probe set, multiple amplification enzyme systems are used, and finally, the most suitable amplification enzyme combination for this gene system is optimized.

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.

The invention discloses a microdroplet type digital PCR (Polymerase Chain Reaction) premixed solution and application thereof in detecting a high-GC target, and belongs to the technical field of gene detection. The microdroplet type digital PCR premixed solution comprises 3 to 5 mM of MgCl2, 60 to 80 mM of KCl, 10 to 30 mM of Tris-HCl with the pH value of 8.3, 150 to 200 nM of dNTP, 0.1 to 0.2 U / l of hot start Taq enzyme, glycerol with the volume fraction of 0.25 to 1 percent, betaine with the volume fraction of 0.25 to 0.75 M, EDTA (Ethylene Diamine Tetraacetic Acid) with the volume fraction of 0.25 to 0.75 mM and formamide with the volume fraction of 0.5 to 2 percent. The premixed liquid has the advantages of good uniformity, high thermal stability, accurate amplification and good Taq enzyme activity maintenance, is suitable for a probe method, provides an efficient universal reagent for a droplet type digital PCR instrument, and realizes high-sensitivity detection of C228T mutation in a TERT gene high GC content (more than 70%) region.

The invention relates to the field of bioengineering, in particular to CRISPR / Cas9 nucleic acid, a recombinant vector, a recombinant engineering bacterium, mRNA and a preparation method thereof, a recombinant protein, a composition, an expression and gene editing method and a product, and the CRISPR / Cas9 nucleic acid provided by the embodiment of the invention comprises a nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5. According to the present invention, by optimizing the codon ratio, the GC content, the sequence repeatability, the RNA secondary structure, the RNA free energy and the like in the DNA sequence, the DNA sequence capable of highly expressing the Cas9 protein version is obtained; the expression quantity of the CRISPR / Cas9 protein provided in the technical scheme is increased by 4-8 times compared with that of a wild type or other contrast, and a similar editing effect can be achieved with a lower dosage, so that the application value of the Cas9 protein in gene editing can be greatly improved.

The invention discloses a multiple PCR primer design method for drug-resistant mutation targeted detection and application thereof, and belongs to the field of biotechnology primer design, the method comprises the following steps: determining a drug-resistant gene of a target species and a detection site thereof; generating a primer set on the basis of one step length based on the target sequence position of the drug-resistant gene mutation site; the candidate primer is subjected to primer attribute evaluation such as TM value, primer length and GC content; meanwhile, a specific sequence is obtained through human-derived sequence non-specific amplification evaluation, dimer evaluation and comparison of a candidate primer and a nt library, the primer coverage degree is evaluated based on a species genome, a conservative primer sequence is obtained, and finally, a final target drug-resistant gene mutation site primer is obtained based on a mutation frequency result. The method is wide in applicability, parallel detection of multiple drug-resistant genes in a single cell can be achieved, and a new strategy is provided for developing design of multiple PCR system primers for drug-resistant mutation targeted detection.

The invention discloses a stable and high-efficiency high-fidelity DNA polymerase mutant and a preparation method thereof, relates to the fields of genetic engineering and minute biology, and aims to modify a high-fidelity DNA polymerase by rationally designing mutation sites and optimizing additives, enhance the thermal stability, extension efficiency and continuous synthesis capability of the high-fidelity DNA polymerase and maintain the high-fidelity characteristic of the high-fidelity DNA polymerase. The optimized DNA polymerase shows significantly improved amplification speed in the PCR amplification process, can efficiently synthesize longer DNA fragments, and is suitable for amplification of complex templates (such as sequences with high GC content or rich secondary structures). According to the method, the synthesis efficiency and accuracy of the long-fragment DNA are remarkably improved, and a more efficient tool is provided for genome sequencing, gene cloning, in-vitro diagnosis and the like.

The application discloses a siRNA sequence design method and an activity prediction device based on deep learning, and relates to the field of artificial intelligence assisted biotechnology. The method comprises the following steps: performing global scanning on a target mRNA, intercepting the target mRNA into RNA sequence fragments according to a predetermined step and a predetermined window width, and enumerating all available candidate siRNA sequence fragments; filtering out sequence fragments with GC content outside a predetermined range in the candidate siRNA sequence fragments, and excluding 15-mer sequences with a frequency greater than a preset threshold in a transcriptome; inputting siRNA sequence data, mRNA sequence and structure data and experimental information into a deep learning model, outputting an activity prediction value, analyzing siRNA sequences with high activity ranking, and outputting potential siRNA sequences. The application can more accurately predict the activity of siRNA in a real experimental scenario and efficiently design siRNA sequences.

The present application relates to a kind of primer group, kit and method for detecting ATXN8OS and ATXN8 gene (CTA·TAG) n And (CTG·CAG) n Trinucleotide repeat number, belong to gene detection technical field.The primer group includes first primer pair and second primer pair;The forward primer sequence of the first primer pair is as shown in SEQ ID NO.1, and the reverse primer sequence is as shown in SEQ ID NO.2;The forward primer sequence of the second primer pair is as shown in SEQ ID NO.1, and the reverse primer sequence is as shown in SEQ ID NO.3.The present application is realized by the design of two primer pairs, complementary verification is realized, detection result is comprehensive and reliable, make up the technical deficiency of one generation sequencing and two generation sequencing in long fragment, high GC content repeat sequence detection, and detection cost is low, detection cycle is short.

The application discloses a DNA storage coding method with local GC content balance, which adopts a four-element sequence coding method for original data, and adopts a splicing method to splice a GC content globally balanced code word set for several times, so that a new four-element code satisfies the GC content local balance, so that the four-element DNA sequence composed of A, T, C and G can be more conveniently corresponded, and the base substitution error and the base insertion and deletion error can be respectively corrected by combining a Hamming code and a VT code. Furthermore, the GC content of the DNA coding can be finally ensured to be globally stable at 40% to 60% by selecting the code word satisfying the GC content in the Hamming code, and the DNA segment in each fixed length sliding window satisfies the GC content stable at 30% to 70%.

The invention belongs to the field of biological information detection, and provides a multiplex PCR (polymerase chain reaction) detection primer group for simultaneously detecting Newcastle disease virus, avian influenza virus, avian coronavirus and Rosella rosea and an application of the multiplex PCR detection primer group for simultaneously detecting Newcastle disease virus, avian influenza virus, avian coronavirus and Rosella rosea. The formation of a primer dimer or non-specific binding with other amplified fragments is prevented; the GC contents of the upstream primer and the downstream primer are similar, and more than three continuous G or C do not appear at the 3'end. The annealing temperatures of the primers are similar, the length difference of the target genes of the amplified fragments is distinguished through electrophoresis, and the length difference of the corresponding target genes among the primer groups is greater than or equal to 170bp; the kit can be used for quadruple PCR detection of the Newcastle disease virus, the avian influenza virus, the avian coronavirus and the Rosella murine, the simultaneous amplification and detection of the four pathogens are successfully realized, the result is accurate and stable, the identification degree is high, and the repeatability is good.

The invention relates to a Klotho messenger RNA (mRNA). Wherein the mRNA has a 5'cap region, a 5 'untranslated region (5'-UTR), a coding region encoding a Klotho polypeptide, a 3 'untranslated region (3'-UTR) and a polyadenosine tail (poly A tail) wherein the Klotho polypeptide comprises the KL1 domain of human Klotho, preferably wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having at least 80% sequence identity to SEQ ID NO: 1, and wherein the coding region encoding the Klotho polypeptide preferably has a GC content of at least 54%.

The application discloses a PTPRQ gene mutation site related to non-syndrome deafness, the mutation site is a novel pathogenic mutation site on a human PTPRQ gene, and is one or more combinations selected from c.4723C>T, c.6040C>T, c.1811T>C, c.5687-6del and c.6560C>T. A specific probe set for detecting a PTPRQ gene mutation, the probe set is a double-stranded DNA probe, is designed according to a PTPRQ gene 45-exon sequence of a transcript NM_001145026 and a flanking region of each 5' end and 3' end of each exon with an extension of 50 bp, contains 169 probes in total, the length of a single probe is 120 bp, a shingled design is adopted, there is an overlapping region between adjacent probes, and the probe density is dynamically adjusted according to the GC content of a target sequence. A detection method of a PTPRQ gene mutation, with peripheral blood or a blood sheet of a to-be-tested person as a sample, extracting genomic DNA, performing targeted capture enrichment by using the specific probe set, combining high-throughput sequencing with standardized bioinformatics analysis, and judging whether the PTPRQ gene mutation exists in the to-be-tested sample.

The application belongs to the technical field of fluorescent quantitative PCR, and provides an enzyme digestion-fluorescent quantitative PCR detection method. The method comprises the following steps: step one, configuring a reaction mixture, wherein the reaction mixture comprises amplification reagents, a restriction endonuclease, Taq DNA polymerase, UDG enzyme, primers and a probe; and step two, setting a reaction program and a reaction system. Non-target templates in a qPCR reaction system are degraded by enzyme digestion of a restriction endonuclease, thereby reducing non-specific amplification; the proportion of target templates is increased, thereby avoiding the interference of non-target templates and improving detection sensitivity and detection stability. In particular, the method can effectively detect high GC content templates, such as TERT gene promoters or CpG island methylation.

The invention discloses a chimeric lysozyme gene mutation analysis method based on genome sequencing data, and relates to the technical field of mutation analysis, the chimeric lysozyme gene mutation analysis method comprises the following steps: carrying out quality control processing on obtained chimeric lysozyme genome sequencing original data to generate a high-quality sequencing read; based on high-quality sequencing reads, the occurrence frequency of k-polymer fragments in each window and a GC variation anomaly sliding window set are obtained, and splicing fracture intervals on the sequencing read set and the coordinate range of splicing breakpoints in the splicing fracture intervals are recognized by combining mutation enrichment regions on a genome. The method comprises the following steps: performing window division on a high-quality sequencing read section, performing statistics on occurrence frequency of a k-polymer fragment and basic group GC content variation, and accurately identifying a splicing fracture interval and a breakpoint coordinate range in combination with genome mutation enrichment regions so as to realize accurate positioning of a genome structure abnormal region.

The invention discloses a universal insertion sequence and application thereof in preparation of multiple PCR template nucleic acid. The invention provides a universal insertion sequence and a method for preparing multiple PCR template nucleic acid based on the universal insertion sequence, and the method comprises the following steps: firstly, designing universal insertion sequences with different GC contents, and distributing a universal insertion sequence with similar GC content for each multiple PCR target amplicon; the method comprises the following steps: firstly, synthesizing a universal insertion sequence primer, then synthesizing a hybrid PCR primer containing the universal insertion sequence primer and a multiplex PCR target amplicon primer, and introducing the multiplex PCR target amplicon into the universal insertion sequence through one-step PCR amplification, so as to obtain a set of artificial chimeric DNA template containing the multiplex PCR primer, and the artificial chimeric DNA template can be used as multiplex PCR template nucleic acid for developing and optimizing a multiplex PCR system.

The present invention relates to a Klotho messenger RNA (mRNA), wherein the mRNA has a 5' CAP region, a 5' untranslated region (5'-UTR), a coding region encoding a Klotho polypeptide, a 3' untranslated region (3'-UTR), and a polyadenosine tail (poly-A tail), wherein the Klotho polypeptide comprises the human Klotho KL1 domain, preferably wherein the coding region encoding the Klotho polypeptide comprises an RNA sequence having at least 80% sequence identity with SEQ ID NO: 1, and wherein the coding region encoding the Klotho polypeptide preferably has a GC content of at least 54%.

The application discloses a quaternary DNA storage method and system based on entropy coding and rotation constraint coding, a storage medium and a terminal. First, compared with traditional binary coding, quaternary coding can improve storage density, thereby storing more data in smaller space. Second, dynamic rotation coding ensures that the DNA sequence meets strict biochemical constraints, including homopolymer length limitation, GC content balance and avoidance of specific harmful sequences, optimizes the synthesis and sequencing process of DNA, and reduces the error rate. In addition, the use of tANS coding provides higher data compression efficiency, further reducing storage costs. These technical innovations not only improve the performance of large-scale data storage, but also enhance the security and accuracy of data decoding, ensuring the integrity of data after long-term storage.

The invention discloses a wheat generic genome 120K liquid phase chip and application thereof. The chip integrates 124 and 265 oligonucleotide probes with the length of 90 bp, the GC content of each probe is 0.3-0.65, the sequence complexity is larger than or equal to 0.9, the annealing temperature (Tm) is larger than or equal to 68 DEG C, the chip does not contain N sites, the single-base tandem repeat number is smaller than or equal to 7, the multi-base tandem repeat number is smaller than or equal to 3, and the number of single-probe matching sites in whole genome alignment does not exceed 10. The probe is designed based on a plurality of mainstream wheat genomes such as IWGSCv2.1, AK58, KN9204 and JING411, and high-coverage and high-polymorphism typing detection of multiple versions of genomes is realized through strict bioinformatics quality control and uniform layout. The chip is suitable for wheat variety typing, molecular assisted breeding, whole genome association analysis and germplasm resource evaluation, and can provide an efficient and accurate technical platform for molecular breeding and genetic improvement of wheat and other gramineous crops.

Provided herein are methods of identifying a subject as having a disease, the method comprising: (a) obtaining a biological sample from the subject, wherein the biological sample comprises cell-free DNA (cfDNA), wherein the cfDNA comprises a plurality of cfDNA fragments; (b) determining an end sequence of a cfDNA fragment of the plurality of cfDNA fragments; (c) determining a level of GC content of the cfDNA fragment; and (d) analyzing the determined end sequence and the level of GC content of the cfDNA fragment, thereby identifying the subject as having the disease by determining a relationship between the determined end sequence and the level of GC content of the cfDNA fragment.

The invention provides an MMLV enzyme mutant as well as a preparation method and application thereof, and relates to the technical field of biology. The MMLV enzyme mutant is obtained by mutation of an amino acid sequence based on a wild type MMLV enzyme amino acid sequence as shown in SEQ ID NO.1, and comprises at least one of amino acid mutations S255K, P315W and L486W. According to the invention, S255K, P315W or L486W mutation is introduced, so that the thermal stability of the MMLV enzyme is obviously improved. Due to the characteristic, reverse transcription can be carried out at high temperature, so that error products are reduced by increasing the specificity of primer combination, a complex secondary structure of RNA is effectively solved, and finally, the reverse transcription efficiency of difficult templates with high GC content and the synthetic ability of full-length cDNA are remarkably improved.