7 results about "Restriction enzyme digestion" patented technology

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 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.

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 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.

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.