14 results about "Restriction enzyme digestion" patented technology

The invention belongs to the technical field of fish genetic identification, discloses a multi-site large yellow croaker wild domestication population identification system based on an agarose gel electrophoresis technology, and particularly discloses a group of InDel molecular markers for large yellow croaker wild and domestication population identification, and the InDel molecular markers comprise InDel11-5053756, InDel20-14004876, InDel3-28345321 and InDel21-17327392. The InDel molecular marker detection primer is used for amplification to obtain a PCR product, agarose gel can be used for detecting strip polymorphism, extra restriction enzyme digestion or complicated polyacrylamide gel electrophoresis operation is not needed, and identification of large yellow croaker wild and domesticated populations can be rapidly and accurately achieved.

This invention relates to an identification method and kit for overlapping PCR with enzyme digestion. The method includes selecting a corresponding restriction endonuclease using N4N5X or N4N5Y as the last three bases of the introduced restriction site; if no corresponding restriction endonuclease is available, selecting a corresponding restriction endonuclease using N5X or N5Y as the last two bases of the introduced restriction site; amplifying to obtain a universal fragment primary PCR product; designing PCR amplification primers based on the restriction endonuclease and universal primers to obtain the primary PCR product of the template to be tested; and performing enzyme digestion and genotyping on the final product of the overlapping PCR amplification. This invention utilizes the DOPCR method to identify SNPs / InDels. Compared with existing methods that create restriction sites, it adds an overlapping PCR step, resulting in significant differences in the digested fragments. It eliminates the need for PAGE electrophoresis; detection can be completed using ordinary electrophoresis, making it low-cost and rapid.

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 disclosure provides methods and compositions for preparation of a nucleic acid library. In some embodiments, the nucleic acids comprise cell-free DNA, including cfDNA that is in need of analysis, such as by sequencing. The methods may comprise restriction enzyme digestion, adapter ligation, and subsequent amplification, and may provide improved approaches for reducing the number adapter dimers produced during the process. In an aspect, a method for preparing a library of nucleic acids may comprise: digesting DNA molecules with restriction enzymes to produce DNA fragments; ligating adapters to the DNA fragments by incubating with ligase to produce a mixture of adapter-ligated DNA fragments and adapter dimers; amplifying the adapter-ligated DNA fragments to produce amplified adapter-ligated DNA fragments; and reducing the quantity of the adapter dimers by differentiating between the junction between an adapter and a DNA fragment, and the junction between an adapter and another adapter.

The application belongs to the technical field of synthetic biology, and discloses a high-purity and high-yield industrial preparation method of a high-molecular-weight polynucleotide (PN). The method uses a plasmid containing a PN base sequence as a template, generates long-chain DNA by using a rolling circle amplification (Rolling Circle Amplification) technology, and then realizes efficient separation of the PN segment by restriction enzyme digestion. The template plasmid is designed, the replication initiation site and the enzyme cutting site are embedded in the corresponding ends of the PN sequence, a single continuous segment seamlessly fused with the PN sequence is formed, 100% theoretical recovery of the PN segment is realized, and the subsequent purification process is significantly simplified. Through the fusion design of the plasmid backbone structure and the PN sequence, the application realizes low-cost and easy-to-scale production of the PN prepared by the rolling circle amplification, and improves the yield and purity of the target product. The process realizes industrial preparation of the high-molecular-weight polynucleotide (PN), and provides a new technical path of safe and reliable, efficient and convenient, and scalable amplification for industrial production of the high-molecular-weight PN.

The present invention belongs to the fields of genetic engineering and edible mushroom breeding technology, and discloses a high-yield, growth-promoting, anti-browning and disease-resistant Agaricus bisporus strain, gene, vector, method and application, and a high-yield, growth-promoting, anti-browning and disease-resistant Agaricus bisporus AbMYB11 overexpression vector. The overexpression vector is obtained by ligating AbMYB11 with the pCAMBIA1303 vector to construct a complete plasmid, followed by restriction enzyme digestion verification and sequencing verification. An Agaricus bisporus strain containing the AbMYB11 overexpression vector. The present invention conducts research at the gene level, improves the resistance quality of Agaricus bisporus and alleviates the situation of insufficient market supply, and provides an Agaricus bisporus strain with growth promotion, anti-browning and strong disease resistance to solve problems such as easy browning and low yield of Agaricus bisporus.

The present invention discloses a chicken NRG4 gene proximal promoter and bidirectional promoter, as well as a construction method and application thereof, belonging to the technical field of genetic engineering. The chicken NRG4 gene proximal promoter is any one of the A1 - A4 sequences with the nucleotide sequences shown in SEQ ID NO: 1 - SEQ ID NO: 4. In the present invention, DNA extracted from chicken whole blood is used as a template, and four kinds of chicken NRG4 gene proximal promoters are obtained by using primers for PCR amplification and restriction enzyme digestion. Among them, the A1 promoter has strong bidirectional promoter activity. The present invention also discloses that the chicken NRG4 gene proximal promoter is regulated by a specific transcription factor C / EBPα, which has potential application value for revealing the transcriptional regulation mechanism of chicken NRG4, cultivating low-abdominal-fat broilers, and revealing the molecular mechanisms behind the occurrence of various human diseases and developing drugs targeting NRG4 expression.

This invention discloses a CAPS molecular marker for enhancing the cold tolerance gene OsSGR in rice, along with its detection and identification methods and applications, belonging to the field of plant molecular breeding technology. The molecular marker consists of two primers, OsSGR-CAPS-F (SEQ ID NO.1) and OsSGR-CAPS-R (SEQ ID NO.2), and can rapidly and accurately distinguish between two OsSGR alleles through PCR amplification, Nhe I restriction enzyme digestion, and electrophoretic analysis. The presence of only a 1038 bp band indicates a cold-sensitive genotype, while the presence of two bands (231 bp and 800 bp) indicates a cold-tolerant genotype. This invention also provides a method for genotype detection using this marker and its application in backcross breeding for selecting cold-tolerant rice lines. This marker system has advantages such as accurate detection, simple operation, and high breeding efficiency, making it suitable for large-scale application.

This application provides a recombinant oncolytic virus carrying a PD-1 / RGMb bispecific antibody, its preparation method, and its application. The preparation method includes the following steps: S1: preparing the plasmid pXN2-M51R-GFP; S2: obtaining the PD-1 / RGMb bispecific antibody sequence; S3: amplification of the Mouse PD-1 / RGMb bispecific antibody; S4: restriction enzyme digestion of the pXN2-M51R-GFP vector; S5: ligation of the target gene to the vector and transformation of the ligation system; S6: packaging of the recombinant vesicular stomatitis virus; S7: infection of Vero with the recombinant vesicular stomatitis virus; S8: resuspension and extraction of the target virus. In this application, the oncolytic virus is engineered using viral reverse genetics technology to design and construct a tandem PD-1 / RGMb bispecific antibody, which is then integrated into the vesicular stomatitis virus genome. This strategy aims to simultaneously block the PD-1 / PD-L1, PD-1 / PD-L2, and PD-L2 / RGMb signaling axes, thereby reshaping the immunosuppressive tumor microenvironment and synergistically enhancing the oncolytic virus-induced antitumor immune response.

The invention provides a universal molecular cloning simulation method based on DNA sequence terminal base complementation. The supported cloning methods are all molecular cloning methods based on DNA sequence terminal base complementation, and include but are not limited to restriction enzyme digestion and linking, Jinmen cloning, Gibson assembly and In-Fusion assembly. The supported linearization mode of the vector and the DNA insertion sequence comprises enzyme digestion linearization, PCR linearization, and enzyme digestion linearization after PCR, and the linear sequence is directly used. The system processes a vector and a DNA insertion sequence according to a selected linearization mode to generate a DNA sequence fragment with a blunt end or a protruding end, and then pretreats the blunt end or the protruding end of the sequence fragment according to a selected cloning method to generate a protruding end which can be used for final connection. And finally, carrying out connection simulation on the connectable tail end with the protrusion, and finally generating a simulation result. The simulation result lists all possible products and possible problems.

The invention relates to the field of gene engineering, in particular to a construction method for an inserted donor fragment. The method comprises the following steps: (1) designing primers according to a genome sequence, and carrying out PCR on a genome; (2) designing a primer according to the plasmid sequence, and carrying out PCR (Polymerase Chain Reaction) on the plasmid or a target cell carrying the plasmid; (3) purifying the upstream and downstream fragments of the to-be-inserted site in the step (1) and the linearized plasmid in the step (2) through a purification kit, and then carrying out Gibsen assembly together; (4) screening target cells transformed from the recombinant plasmids to obtain successfully recombined transformants; (5) carrying out PCR (Polymerase Chain Reaction) amplification on the transformant obtained in the step (4) by designing a primer, amplifying a DNA fragment to be inserted by designing the primer, purifying by a purification kit, and then carrying out Gibner assembly; (6) transforming the fragment in the step (5) into a target cell, and screening to obtain a transformant carrying the donor fragment; and (7) carrying out PCR (Polymerase Chain Reaction) or enzyme digestion on the transformant in the step (6) to obtain a donor fragment. The method provided by the invention can effectively reduce the number of times of gene amplification and transformation, reduce the probability of mutation, shorten the construction time of donor fragments, reduce the labor intensity of experimenters and save reagents.

The invention belongs to the technical field of synthetic biology, and particularly relates to an industrial preparation method of high molecular weight polynucleotide (PN). According to the method, a plasmid containing a PN base sequence is used as a template, long-chain DNA is generated by using a rolling circle amplification technology, and then efficient separation of PN fragments is realized through restriction enzyme digestion. The plasmid is subjected to optimization design, a restriction enzyme cutting site is introduced into a replication start site and a selection marker gene, after enzyme cutting treatment, a plasmid skeleton is degraded into small fragments smaller than 250 bp, and efficient separation of a target PN fragment is achieved by means of molecular weight difference. Through optimization design and large-scale preparation of a plasmid template, low-cost and easy-to-amplify production of PN prepared by rolling circle amplification is realized, and meanwhile, the length and sequence of a final PN product can be precisely designed, regulated and controlled. Particularly, a specific enzyme cutting site is introduced into a plasmid template, so that the subsequent precise removal of a plasmid skeleton sequence is facilitated, and the purity of a target product is remarkably improved. According to the process, the industrial preparation of the high molecular weight polynucleotide (PN) greater than 1500 bp is successfully realized, and a safe, efficient, convenient and large-scale brand new path is provided for the production of the high molecular weight PN.

Embodiments of the disclosure include methods of producing libraries of nucleic acids, including genomes and / or transcriptomes, including for single cells and / or subcellular structures. In specific embodiments, a double stranded DNA library is produced upon fragmentation by a Tn5 transposase having restriction enzyme site(s), digestion by the restriction enzyme, and ligation of adaptors to the ends.

The application discloses a corn lodging-resistant molecular marker and application thereof. The molecular marker SNP-1642 is a base substitution of "C / T" at the 1642th bp in the Zma-miR528b gene promoter region, the miR528 has a higher expression amount in the inbred line with the SNP-1642 genotype of T, and is accompanied by a higher lodging index, and the miR528 has a lower expression amount in the inbred line with the genotype of C, and is accompanied by a lower lodging index. Based on the single nucleotide polymorphism at the site, a restriction enzyme digestion amplification polymorphic sequence is developed. The molecular marker provided by the application can screen new corn varieties resistant to lodging through different haplotype nucleotide sequences, and has important application value for improving the lodging resistance of corn.