86 results about "Restriction enzyme" patented technology

The invention belongs to the field of molecular biological detection, and particularly relates to an FBXO32 gene SNP (Single Nucleotide Polymorphism) marker for rapidly detecting economic traits of Tibetan chicken as well as a detection method and application. It is found for the first time that G / A mutation at the 8916th site of the Tibetan chicken FBXO32 gene is closely related to whether the gene has an Hpa II restriction enzyme cutting site or not, and is closely related to main economic traits of Tibetan chicken. Based on the new discovery and the SNP site, the invention provides a method for rapidly detecting the SNP site and screening economic characters of Tibetan chicken. The method is simple to operate, convenient and fast, low in cost and reliable in result.

The invention belongs to the field of molecular biological detection, and particularly relates to a PLS3 gene SNP marker for rapidly detecting economic traits of Tibetan chicken, a detection method and application. It is found for the first time that G / A mutation at the 28469th site of the Tibetan chicken PLS3 gene is closely related to whether the gene has a BstZ17 I restriction enzyme cutting site or not, and is closely related to main economic traits of Tibetan chicken. Based on the new discovery and the SNP site, the invention provides a method for rapidly detecting the SNP site and screening economic characters of Tibetan chicken. The method is simple to operate, convenient and fast, low in cost and reliable in result.

The invention discloses a whole genome methylation detection method based on MeDIP-MSRE, and belongs to the technical field of epigenetics detection. According to the method, methylation immunoprecipitation sequencing and a methylation sensitive restriction enzyme technology are innovatively combined, firstly, a methylation specific antibody is used for conducting immunoprecipitation on sample DNA, and whole genome methylation fragments are enriched; then carrying out enzyme digestion on the enriched product by adopting methylation sensitive restriction enzyme, specifically removing an unmethylated DNA region, and reserving a complete methylation sequence; and finally, constructing a methylation map through high-throughput sequencing. According to the method, traditional hydrosulfite chemical conversion is not needed, DNA damage and base conversion deviation caused by the traditional hydrosulfite chemical conversion are avoided, meanwhile, high sensitivity of MeDIP and high specificity of methylation sensitive restriction endonuclease are fused, and the fidelity, sensitivity and specificity of detection are remarkably improved.

The invention relates to the technical field of molecular biology, in particular to a methylation marker for diagnosis of diabetic nephropathy. Specifically, the methylation marker comprises a P site of a CAT gene promoter region and / or an E site of a CAT gene exon 1 region. Based on a detection system combining methylation sensitive restriction enzyme with quantitative PCR, the kit has the advantages of simplicity and convenience in operation, good repeatability, accuracy in quantification and easiness in standardization, and is suitable for low-invasive samples such as peripheral blood. By effectively distinguishing pure type 2 diabetes patients from diabetic nephropathy combined patients, a reliable technical means is provided for risk stratification, early intervention and dynamic monitoring of diabetic people, and the method has good clinical application prospects and popularization value.

Methods for the processing and analysis of blood samples obtained with blood collection tubes that reduce contamination of cfDNA by genomic DNA but that inhibit digestion by methylation-sensitive and / or methylation-dependent restriction enzymes.

The invention discloses a dCAPS molecular marker primer linked with a brassica napus saline-alkaline tolerance gene BnaC03.RBGB3 and application of the dCAPS molecular marker primer. The primer comprises a forward primer and a reverse primer, and through PCR amplification and specific restriction enzyme digestion treatment, two genotypes of high resistance and high sensitivity of brassica napus saline-alkaline tolerance can be distinguished on agarose gel electrophoresis; the high-resistance genotype is represented by two electrophoretic bands (195bp and 23bp), and the high-sensitivity genotype is a band which cannot be digested by enzyme. The method disclosed by the invention is simple and convenient to operate and reliable in result, can be used for rapidly screening and identifying the saline-alkaline-tolerant variety of the brassica napus, and provides a powerful tool for breeding the saline-alkaline-tolerant variety of the brassica napus. By implementing the technology, not only is the breeding efficiency improved, but also a new thought and strategy are provided for molecular marker-assisted breeding of saline-alkaline tolerance of crops, and development and application of a saline-alkaline tolerance breeding technology of brassica napus and even other crops are promoted.

The invention discloses a dCAPS molecular marker for identifying sweet potato pulp color characters and application of the dCAPS molecular marker, and belongs to the technical field of sweet potato breeding. According to the dCAPS molecular marker provided by the invention, on the basis of SNP variation sites, mismatched bases are introduced to form restriction endonuclease restriction enzyme cutting sites, the sweet potato pulp color can be judged by utilizing PCR-restriction enzyme cutting-polyacrylamide gel electrophoretic separation, and then sweet potato varieties with high and low carotenoid contents can be identified according to the sweet potato pulp color. The method has important guiding significance and application value for assisting sweet potato carotenoid content related molecular breeding research.

The present disclosure provides a method for detecting the methylation and mutation status of a DNA sample, specifically, a method for simultaneously detecting the methylation and mutation status of DNA molecules in a sample, the method comprising treating the sample with a methylation-sensitive restriction enzyme, performing hybrid capture on amplification products of the restriction enzyme-treated sample and a sample that has not been subjected to enzyme cleavage treatment with a probe set including a mutation capture probe and a methylation capture probe, and sequencing the captured products.

[Problem] To provide: an adapter for binding to polynucleotide fragments that is used for preparing a genome-wide gRNA library for genome editing systems; a gRNA library that is obtained by using the adapter; and uses of the gRNA library. [Solution] An adapter having a structure described in A). A) A structure having i) to iii). i) A polynucleotide chain having sequences i)-1 to i)-3. i)-1: Sequence corresponding to the forward primer. i)-2: Sequence of the recognition site of a restriction enzyme capable of excising a gRNA spacer sequence from the 5' end of a polynucleotide fragment bound to the adapter. i)-3: Sequence of the recognition site of a restriction enzyme capable of cleaving an adapter having the structure of A) from the polynucleotide fragment. ii) A polynucleotide chain having the sequence complementary to i). iii) A single-stranded polynucleotide chain for formation of a loop connecting the 5' end of i) and the 3' end of ii) of the double strand resulting from the binding of i) and ii).

The invention belongs to the technical field of genetic engineering, and particularly relates to a universal bacterial artificial chromosome recombinant virus transfer vector and a construction method thereof, the transfer vector comprises a BAC amplification vector with pUC19-loxp-gpt-IRES-EGFP as a basic skeleton and pUC19-BAC as a functional sequence; according to the universal bacterial artificial chromosome recombinant virus transfer vector and the construction method thereof, a drug screening target gene reading frame and an EGFP fluorescent protein gene reading frame are connected through an IRES double-expression connection sequence, and double screening can be carried out; in the BAC construction process, screening genes and functional sequences are respectively constructed to a high-copy T vector, so that sufficient plasmid DNA can be conveniently copied and prepared. And through a proper restriction enzyme cutting site, introduction of various homologous arms is facilitated, the alignment is wide, simultaneous operation of various genomic sequences is facilitated, and the research efficiency is improved.

The invention provides a method and a device for simulating a pulsed field gel electrophoresis band based on a sequencing technology. The method for simulating the pulsed field gel electrophoresis band based on the sequencing technology comprises the following steps: acquiring nanopore sequencing data of a biological sample to be analyzed, and assembling based on the nanopore sequencing data to obtain a genome sequence; according to the sequence characteristics of the restriction endonuclease, identifying a restriction enzyme cutting site corresponding to the restriction endonuclease from the genome sequence; when the restriction enzyme cutting site is the restriction enzyme cutting site subjected to methylation modification, removing the restriction enzyme cutting site to obtain a residual restriction enzyme cutting site; calculating the distance difference between two adjacent restriction enzyme cutting sites according to all residual restriction enzyme cutting sites, and taking the distance difference as the molecular weight of a simulated band; aiming at the molecular weight of each simulated strip, determining a strip position corresponding to the molecular weight; and drawing a simulated pulse field gel electrophoresis band result according to the position of each band and the molecular weight corresponding to the band position so as to accurately simulate the band result.

The present disclosure provides methods and compositions for preparing DNA molecules useful for determining the methylation status of selected regions of a DNA molecule, which utilize methylation-sensitive restriction enzymes and typically involve the amplification or selective enrichment of one or more target regions.

The invention discloses a high-efficiency expression method of porcine interleukin 22 (pIL-22) and application of the pIL-22 in resisting PEDV (porcine epidemic diarrhea virus) infection, and belongs to the technical field of biology. The high-efficiency expression method comprises the following steps: inserting a swine-derived interleukin 22 gene (SEQ ID NO: 1) into a pcDNA3.1 (+) vector restriction enzyme cutting site BamH I / Xho I to obtain a recombinant vector, mixing the recombinant vector and a transfection reagent, transfecting HEK-293 cells, collecting a cell suspension, ultrasonically crushing, filtering with a filter membrane to obtain a target protein, and finally purifying with a Protein A chromatographic column to obtain the swine-derived interleukin 22. (1) by adopting a mammalian cell expression system and a culture mode of suspension cells, not only can high-activity pIL-22 be expressed, but also the expression efficiency of the pIL-22 is greatly improved, and important guarantee and reference basis are provided for developing novel anti-PEDV drugs;

The invention belongs to the technical field of polypeptide preparation and biological medicine, and particularly relates to a preparation method of Jingzhaotoxin polypeptide IX (JZTX-IX) and application of Jingzhaotoxin polypeptide IX in treatment of diabetes mellitus. A chilobrachys jingzhao toxin polypeptide IX coding gene, a 6 * His tag nucleotide sequence, a thrombin restriction enzyme cutting site nucleotide sequence and an MBP hydrotropy tag nucleotide sequence are connected to a prokaryotic expression vector for prokaryotic expression, and the expressed polypeptide is purified to obtain the chilobrachys jingzhao toxin polypeptide IX protein. The target polypeptide with high purity, complete disulfide bond folding and uniform conformation is obtained, so that the high purity and biological activity of the target polypeptide are ensured. On the basis, the treatment activity of the JZTX-IX on diabetes mellitus especially type II diabetes mellitus is found and verified, and the JZTX-IX can remarkably reduce fasting blood glucose of mice with the type II diabetes mellitus, improve glucose tolerance and insulin sensitivity, relieve polydipsia and polyuria symptoms and promote insulin secretion and islet cell proliferation.

To provide a preparation method of a unit DNA composition in which the mole number of a plurality of unit DNA are arrayed better, and a manufacturing method of a DNA connected body.SOLUTION: A preparation method of a unit DNA composition has: a process of preparing solution including a plurality of unit DNA, to which an additional sequence is connected, for each kind of the unit DNA; a process of, after preparation of each solution, measuring the concentration of the unit DNA in each solution in the state where an additional sequence is connected to the unit DNA, dispensing each solution on the basis of its result, and making the mole numbers of the unit DNA in each solution become closer to the same with each other. A manufacturing method of a DNA connected body has: a process of preparing a unit DNA composition; a process of preparing a vector DNA; a process of removing each additional sequence from the unit DNA in which an additional sequence in solution after preparation is connected by using restriction enzyme; and a process of connecting the vector DNA and each unit DNA with each other after the removal process.SELECTED DRAWING: None

The application provides application of a CAPS molecular marker GW18 in identification of foxtail millet (Setaria italica) resistance to rice blast, and belongs to the technical field of molecular marker breeding. The application provides the application of the CAPS molecular marker GW18 in identification of the foxtail millet resistance to the rice blast, and G / T polymorphism exists at 34523782 bp of the 6th chromosome of the foxtail millet; when the polymorphism is G, the resistance to the rice blast cannot be recognized by a restriction enzyme, and is identified as the susceptibility to the rice blast; when the polymorphism site is T, the GW18 target sequence can be recognized by the restriction enzyme and is cut into two segments, and is identified as the resistance to the rice blast. The marker has a high co-separation rate with the disease resistance, a rapid, accurate and low-cost detection method, and can be used for rapid identification of foxtail millet germplasm resources resistant to the rice blast and molecular marker assisted breeding, and has important significance for improving breeding efficiency and guaranteeing safe production of the foxtail millet.

Various compositions and methods are disclosed in which a plurality of restriction enzymes, including methylation-sensitive and / or methylation-dependent restriction enzymes, are used for the analysis of cfDNA. Useful combinations can be inactivated by heating to 65° C., ideally for longer than 15 minutes. Restriction digestion of cfDNA can occur for 11 hours or less. Digestion may be followed by amplification and / or sequencing steps.

This application relates to a method and kit for the specific degradation of library contaminants based on a methylation-sensitive restriction endonuclease. The method includes: determining the recognition site of the methylation-sensitive restriction endonuclease, digesting library contaminants, fragment purification, verification, and quality control. In the library contaminant digestion step, the library reaction system is incubated at 35°C–39°C for 0.5–1.5 hours, followed by incubation at 60°C–70°C for 15–25 minutes to obtain the digested DNA library reaction system. This method selectively removes unmethylated DNA while having almost no effect on highly methylated DNA, avoiding non-specific degradation and effectively improving the purity of the target fragment.

The present invention discloses a composition, a kit and their applications for detecting lung cancer. The composition includes detection reagents for detecting the methylation status of one or more of 17 genes such as SIX2 and PANTR1. The detection reagents contain multiplex PCR pre-amplification primers, real-time fluorescence quantitative PCR primers and probes, and primers and probes for the internal reference ACTB, and are designed by nested PCR, and the primer sequences are SEQ ID No.1-90. The kit contains the above composition and also contains methylation-sensitive restriction enzymes and Exonuclease I. Among them, the real-time fluorescence quantitative PCR primers and probes for 17 genes and ACTB are分装 into 6 reaction tubes. The present invention combines methylation-sensitive restriction enzyme treatment, multiplex PCR pre-amplification and fluorescence quantitative PCR techniques to achieve high-sensitivity and high-specificity detection of lung cancer. The combined detection of 17 genes has an AUC of 0.91, is easy to operate, has low cost, is suitable for early screening and auxiliary diagnosis of lung cancer, and has good clinical application prospects. It should be noted that the word "分装" in the original text seems to be a misspelling. It should probably be "separately packaged" or something similar. The translation above keeps the original text as it is for that word.

Molecular cloning method using Type IIS restriction enzymes The invention is addressed to a cloning method for obtaining a destination nucleic acid vector comprising "n" inserts, wherein "n" is an integer of at least 1, and each insert comprises a sequence of interest "S" and, wherein the method comprises contacting in a reaction mixture: (i) a receiving backbone, wherein: (a) one end of the receiving backbone comprises a palindromic overhang and the other end comprises a non-palindromic overhang, or (b) each end of the receiving backbone comprises a different palindromic overhang; (ii) the n inserts, wherein each insert comprises two different overhangs, wherein the overhangs comprised in the n inserts are different overhangs, and each one is complementary only to another overhang comprised in another insert or to an overhang of the receiving backbone, and is non-palindromic, except the insert's overhang(s) complementary to the palindromic overhang(s) of the receiving backbone, and (iii) a DNA ligase; wherein the molar ratio of the receiving backbone and the insert(s) comprising a palindromic overhang is from 1:0.1 to 1:1.9 (receiving backbone:insert), and the molar ratio of the receiving backbone and the remaining inserts is from 1:1.1 to 1:3 (receiving backbone:insert). In a particular embodiment, at least one of the n inserts and the receiving backbone are obtained upon digestion of one or more polynucleotides and a receiving vector with Type IIS restriction enzyme(s).

The present application relates to a human BMP3 and NDRG4 gene methylation detection kit, the kit comprises a restriction enzyme reaction solution, the restriction enzyme reaction solution comprises a restriction enzyme buffer, a methylation-dependent restriction enzyme, and further comprises a fluorescent PCR reaction solution of primers and a partial double-stranded linear DNA probe designed for a CpG island of a BMP3 gene and / or a NDRG4 gene. The detection kit can realize the sequential restriction enzyme reaction and fluorescent PCR reaction in one tube to detect the methylation state of the BMP3 and NDRG4 genes, and has the characteristics of simple and rapid operation, high sensitivity, good specificity and easy automation.

The invention provides a site-specific mutagenesis vector and a use method thereof, and relates to the field of gene engineering in the biotechnological pharmaceutical industry, the vector contains BbsI restriction enzyme cutting sites except for a polyclone region, and site-specific mutagenesis comprises the steps of carrying out base mutation on the BbsI restriction enzyme cutting sites, removing the BbsI sites and keeping amino acid unchanged. According to the invention, a Golden gate method is adopted to construct the multi-copy vector, and a plurality of copies are directly assembled on a system in one step, so that the time is saved, and the tedious steps of multiple times of enzyme digestion and connection are omitted. According to the invention, a multi-copy vector is constructed in vitro, the copy number of the antibacterial peptide is increased, simultaneous expression of each unit is realized, and the expression quantity is improved. Meanwhile, the antibacterial peptide genes are connected in series by adopting a serial connection technology of the antibacterial peptide genes, so that the expression quantity of the antibacterial peptide is improved on the DNA level.

The present disclosure relates to a method of determining the presence of at least one messenger RNA (mRNA) molecule in a composition, the method comprising: (a) reverse transcription of the at least one mRNA molecule to obtain a template comprising at least one first strand of complementary DNA (cDNA); (b) generating at least one double-stranded cDNA molecule from the template; (c) amplifying the at least one double-stranded cDNA molecule; (d) digesting the at least one amplified double-stranded cDNA molecule with at least one restriction enzyme to obtain a test cDNA fragment; (e) separating the test cDNA fragments so as to form a test cDNA fragment map; and (f) comparing the test cDNA fragment map to a control cDNA fragment map wherein the at least one mRNA molecule is present in the composition when the test cDNA fragment map comprises the control cDNA fragment map. Also disclosed are methods of quantifying and / or assessing the integrity of at least one mRNA molecule in a composition, processes of making a composition comprising at least one mRNA molecule and a primer pair for use in the methods of the invention.

To provide a method for efficiently and accurately distinguishing two similar diseases that differ in the presence or absence of a mutant allele in the same gene region.SOLUTION: A method for distinguishing between a genetic disease caused by a codon mutation in a gene and another disease includes the steps of: performing an amplification reaction to amplify a genetic region containing the codon, the amplification reaction including adding a DNA primer for amplifying the genetic region containing the codon and a PNA that inhibits the amplification of a normal allele containing the codon; and treating a product obtained by the amplification reaction with a restriction enzyme that specifically recognizes and cleaves either a base sequence containing the codon with the mutation or a base sequence containing the codon without the mutation, thereby obtaining a restriction enzyme-treated product.SELECTED DRAWING: Figure 3

The present invention relates to methods for the detection of nucleic acids of defined sequence and kits and devices for use in said methods. The methods employ restriction enzymes. polymerase and oligonucleotide primers to produce an amplification product in the presence of a target nucleic acid. which is contacted with oligonucleotide probes to produce a detector product.

The invention discloses a molecular marker for identifying an excellent haplotype of a corn ZmTAR4 gene and application of the molecular marker, and belongs to the technical field of plant molecular breeding and genetic detection. Specifically, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, and a G / A polymorphic site exists at the 29bp position of the sequence. The invention further develops a primer pair for detecting the molecular marker, a detection product containing the primer pair is prepared, PCR amplification is carried out on corn genome DNA through the primer pair to obtain an amplification product, the amplification product is subjected to enzyme digestion through restriction enzyme, and the excellent haplotypes of the corn ZmTAR4 gene are distinguished according to the obtained enzyme digestion product. The molecular marker is reasonable in design, simple and convenient to operate and accurate in interpretation, is suitable for corn germplasm resource evaluation and molecular marker-assisted breeding, and has important application value in breeding practice for improving corn genetic transformation efficiency.

The invention relates to the field of cell genetic engineering, and discloses a high-sensitivity embryo chromatin analysis method which comprises the following steps: S1, puncturing an embryo zona pellucida through a micromanipulation instrument, sucking 5-8 trophoblast cells, and immersing a sample into 4 DEG C chromatin stabilizing liquid containing 1mM of ethylenediamine tetraacetic acid and bovine serum albumin within 8 seconds after the sample is obtained; s2, processing the sample by adopting a gradient fixation method; s3, sequentially carrying out low-intensity ultrasonication and restriction enzyme combined treatment; s4, carrying out specific labeling by using a double-labeling probe system; s5, implementing two-stage signal amplification in the micro-fluidic chip; and S6, carrying out three-dimensional space conformation analysis by adopting a super-resolution microscope. A silicon nitride microscopic needle penetrates through the zona pellucida at a puncture angle of 75-85 degrees, and the suction speed is controlled to be 0.5 mu L / s in combination with a PID closed loop, so that the DNA fragmentation rate can be reduced to 2.1 + / -0.3% (the proportion of 5kb fragments is 98.5%), and the embryo survival rate is gt; the H3K9 acetylation modification level is improved by 5.3 times, and the chromatin accessibility is improved.

The application provides a Septin9 methylation detection composition and application thereof, and the composition comprises a methylation-dependent restriction enzyme, a capture oligonucleotide, a universal primer and a Septin9 methylation-specific primer; the capture oligonucleotide comprises a first universal sequence, a folding sequence and a binding capture sequence from 5' end to 3' end in sequence; the folding sequence is at least partially identical to a 5' end sequence of a Septin9 methylation site after the methylation-dependent restriction enzyme is cut; and the binding capture sequence is specifically bound to a fragment region where the detected Septin9 methylation site is located. The application is based on a methylation-dependent restriction enzyme and a universal primer fluorescence quantitative PCR technology, does not need bisulfite conversion, and realizes accurate and specific Septin9 methylation detection.

The invention provides a restriction enzyme combination and application thereof. Wherein the restriction endonucleases comprise a combination of restriction endonucleases EcoRV and restriction endonucleases Dral, the combination of the restriction endonucleases can effectively enrich cfDNA fragments where the lung cancer related mutation sites are located, so that the sensitivity of downstream analysis is improved, the mutation detection rate of the lung cancer related mutation sites reaches 95%, and the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result is accurate. The method can be applied to various clinical and research fields, including tumor diagnosis, prenatal screening and disease monitoring.

The invention discloses a composition for detecting spinal muscular atrophy typing based on nucleic acid mass spectrometry and application of the composition. The invention relates to the field of gene detection, and provides a composition for detecting spinal muscular atrophy typing based on nucleic acid mass spectrometry, which comprises a target gene primer pair, a reference gene primer pair and an IIS type restriction enzyme. The composition can be used for detecting spinal muscular atrophy typing based on nucleic acid mass spectrometry, and the traditional technical method of nucleic acid mass spectrometry is improved, so that nucleic acid mass spectrometry detection does not need the steps of SAP treatment and single base extension reaction, the reaction time is shortened, the result is accurate and reliable, the steps are simple, and the cost is greatly reduced. The invention is of great significance to preparation of screening products suitable for large-scale screening of spinal muscular atrophy.