32 results about "Restriction Enzyme Cleavage Site" patented technology

A support for fixing a nucleotide which contributes to the efficient clarification of DNA without damaging the terminal parts of the DNA and, therefore, is useful in the fields of molecular biology, biochemistry and the like. This support for fixing a nucleotide is a chemically modified substrate on which oligonucleotides are immobilized characterized in that the oligonucleotides have a restriction enzyme cleavage site. This support is produced by chemically modifying the substrate, immobilizing a single-stranded oligonucleotide thereon, then hybridizing the single-stranded oligonucleotide with another single-stranded oligonucleotide having a base sequence complementary thereto, and ligating an oligonucleotide having a restriction enzyme site at the end thereof.

The present invention provides a rabies virus CTN strain whole genome cDNA infectious clone, as well as a method of using reverse genetics technology to save the active rabies virus. The present inventive method includes: using the single restriction enzyme sites in the CTN strain of rabies virus full-length genome to divide the full-length virus into four segments to amplify, simultaneously using the green fluorescent protein gene to substitute 423bp base at the pseudo-gene point, cloning in the pVAX-R expression vector, to construct a recombinant full-length plasmid of the virus genome; performing PCR amplification of nucleoprotein, phosphoprotein, glycoprotein and transcription large protein gene sequence of the CTN strain of the rabies virus, and cloning the amplified fragment into a vector pVAX1, to constitute four auxiliary plasmids in the reverse heredity system; and then using the five plasmids to jointly transfect cells, to rescue the rabies virus. The present invention successfully saves the rabies virus, and has a great significance in the study of the pathogenic mechanism of rabies virus, development of new vaccines, viral vectors and the like areas.

The invention provides the preparation and use of a drug overexpressing PCNP gene. The medicine includes a plasmid overexpressing PCNP gene and PCNP protein obtained by overexpressing PCNP gene. The construction method of the plasmid overexpressing PCNP gene, which comprises the following steps: according to the PCNP gene sequence, design primers, PCR amplify the target gene; carry out restriction enzyme cutting site Xho I and Kpn I in the GV230 vector. , to form a linearized GV230 vector; the target gene is connected with the linearized GV230 vector to construct a GV230-PCNP vector, which is a plasmid that overexpresses the PCNP gene. When the drug overexpressing PCNP gene is used in human neuroblastoma, the overexpression of PCNP gene in human neuroblastoma can promote cell apoptosis, inhibit cell growth, inhibit cell migration, and inhibit the growth of human neuroblastoma. grow.

The invention discloses a PCR (polymerase chain reaction) method for eliminating genes, which can eliminate non-target genes in a mixed gene population. The key points of the technology disclosed by the invention are as follows: making or artificially synthesizing the non-target genes into short-sequence gene eliminating primers, performing enzyme cutting on the mixed gene population containing target genes, adding a restriction enzyme cutting site with a base pair mismatch and a joint with Poly (dA), purifying, using the mixed gene population as a template, restoring the correct enzyme cutting site through the gene eliminating primers and annealing extension of the template in the PCR, and then using the heat-resistant restriction enzyme cutting site to eliminate the joint of the non-target genes so as to prevent amplification of the non-target genes. The reaction is cycled by utilizing a PCR instrument: 30s at the temperature of 94 DEG C, 40s at the temperature of 60 DEG C, 8min at the temperature of 75 DEG C, 12-18 cycles are performed for enriching the target genes, and then the conventional PCR is adopted for further amplifying the target genes. The method disclosed by the invention has the advantages of high efficiency, high speed, simplicity and convenience in operation, low cost, high repeatability, good separation effect and low false positive rate.

The invention relates to an ultra-binary vector as well as a construction method and an application thereof. The ultra-binary vector is characterized in that a framework vector of the ultra-binary vector is pCMBIA1301-hpt<->, and Xba I-RB-LB-Sal I is inserted between restriction enzyme cutting sites Xba I and Sal I of the framework vector, thereby forming two T-DNA (transferred-deoxyribose Nucleic Acid) regions together with original left and right border sequences; and hpt II is connected into a T-DNA2 region through the restriction enzyme cutting sites, so as to acquire the ultra-binary vector pDT1301 containing the hpt II. The pDT1301 disclosed by the invention contains the hpt II and is convenient for screening callus-resistant and transgenic regenerated plants; and exogenous target genes of the hpt II are not in the same T-DNA region, so that the self-fertilization and separation of transgenic descendants are facilitated, the safe transgenic plants which do not contain selective marker genes are acquired, and obstacles for the safe release of the transgenic plants are cleared.

The present invention provides PCR-RFLP primers and methods for identifying Mycoplasma mycoplasma goat subspecies and other members of Mycoplasma mycoplasma cluster, the upstream primer P1: 5'-ACAAAAAATATTAGCATTCAAACTT-3', the downstream primer P2: 5'-GGTGTTATTTTTGATTTAG ATGGAG-3' , the method is to use the differences in the enzyme cutting sites contained in the pgmb protein gene coding region of mycoplasma goat subspecies and other members of the mycoplasma cluster to distinguish, including DNA extraction, PCR amplification to obtain the corresponding target fragment RFLP analysis was performed after HindⅢ digestion. The identification method of the invention has simple operation and high accuracy.

The invention provides a method for detecting kanamycin. The kanamycin is detected by using double restriction enzyme cleavage sites and color change caused by G-tetrad. The preparation method specifically comprises the following steps: carrying out specific binding on a hair pin and kanamycin; adding a color developing agent into a homogeneous phase reaction mixed liquor for display detection. According to the method, high specific detection for the target object kanamycin is realized by using the oxidability of the G-tetrad; the effect of signal amplification is reached by using nucleic acid tool enzymes.

The invention discloses a method for detecting the variation and methylation of tumor-specific genes in ctDNA. The method can simultaneously detect the variation of tumor-specific genes in ctDNA (including point mutations, insertion-deletion mutations, and HBV integration) in one sample. and / or methylation method, not only the sample size requirement is low, but also the MC library prepared by this method can support 10‑20 follow-up detections, and the results of each detection can represent all original ctDNA samples The mutation status and the methylation modification of the region covered by the restriction site will not cause a decrease in sensitivity and specificity. The invention has important clinical significance for early tumor screening, disease tracking, curative effect evaluation, prognosis prediction, etc., and has great application value.

The invention relates to a drug conjugate for targeted traceless release, a preparation method and application thereof. Specifically disclosed is a targeted traceless release drug conjugate, the structure of which is T-L-D, T represents a targeting group, L represents a linker, and D represents an active ingredient, wherein T is selected from RGD peptide, cyclic RGD Peptides, RGD peptide derivatives, RGD cyclic peptide derivatives, folic acid, membrane-penetrating peptides, nucleic acid aptamers, fluorescent dyes; D is a pharmaceutical active ingredient with a tertiary amino group, preferably coyba peptide A, coyba peptide A Derivatives; L is A-BC-E, BC represents the enzyme cleavage site. The present invention uses cyclic RGD which has targeting effect and is easy to synthesize and modify instead of expensive and difficult-to-synthesize antibody as the targeting molecule, which is simpler to prepare and achieves high specific targeting effect. Under the action, through the self-elimination of p-aminobenzyl quaternary ammonium salt, the traceless release of the active ingredient of the drug is realized.

The invention discloses an SPAR (single primer amplification reaction) based DNA molecular marking method, which belongs to the field of biotechnology. The method comprises the following steps: designing and synthesizing a single primer, extracting a sample genomic DNA as a template, carrying out PCR amplification in a conventional PCR reaction system and a conventional reaction procedure by using the single primer and the sample DNA template, and after an amplified product is subjected to agarose gel electrophoresis separation, detecting the polymorphism between two binding sites in a genomic gene exon area, wherein the sequence length of the single primer is 17 bp, six base filling sequences at a 5' terminal are AT-base-rich restriction enzyme cleavage site sequences, a mid-core area sequence is randomly composed of 8 GC bases, and 3 selective bases are arranged at the last 3' terminal. The primer in the invention is good in universality, and the DNA molecular marking method disclosed by the invention has the advantages of simple operation, high stability, good repeatability, number abundance, high specificity, high polymorphism, reliable results, and the like.

The invention discloses a detection MTHFR A method and a kit for genotyping polymorphic sites of gene rs1801131. The present invention detects based on the PCR-RFLP method MTHFR In the process of gene rs1801131 polymorphic site genotype, a low-cost endonuclease was selected, and a unique "internal quality control" mechanism was introduced at the same time, that is, an internal control enzyme cutting site was introduced into the PCR product, which can The incomplete digestion can be identified according to the enzyme digestion map, and the interference of residual PCR products or intermediate products of digestion can be eliminated, and the genotype can be accurately judged according to the characteristic bands, which greatly improves the accuracy of the detection results.