12 results about "Dna template" patented technology

The present application relates to the application of Klenow mutant fragments in the synthesis of nucleic acids, and belongs to the technical field of biological modification and synthesis.The present application provides the application of Klenow mutant fragments with an amino acid sequence as shown in SEQ ID NO.8 or SEQ ID NO.14 in the synthesis of nucleic acids.The mutant protein F762A expressed by the Klenow mutant fragments of the present application can efficiently synthesize deoxyribonucleic acid, ribonucleic acid, deoxyribonucleic acid analogs and ribonucleic acid analogs with different lengths of DNA templates and DNA / RNA primers at 37 DEG C, and has a wide synthesis range and universality.

Provided are DNA library preparation methods and compositions that duplicate a target nucleic acid sequence. A target DNA template including the target sequence is circularized via an end adapter to form a circular construct, which is bidirectionally extended by a polymerase-mediated extension that is initiated at nick sites of the end adapter. Following polymerase-mediated extension, a double-length DNA template is formed that includes two copies of the target DNA template (and hence two copies of the target sequence). Each strand of the double-length DNA template includes a parental polynucleotide strand joined to a newly synthesized daughter strand copy of the parental polynucleotide strand. Predetermined sequences can be included in the double-length DNA template, such a primer sequences, unique molecule identifiers, and sequence indexes. Sequencing of the double-length DNA template can reveal genetic / epigenetic information associated with the target sequence. Also provided are methods to create asymmetric and multi-length DNA template constructs.

This invention relates to a catalyst, specifically a sub-10 nm catalyst and its self-assembly preparation method and application, comprising the following steps: pretreating DNA-modified Fe3O4 particles to remove free DNA strands; mixing scaffold DNA, short-chain DNA, and extended-chain DNA in a buffer solution and annealing to self-assemble into a DNA template; mixing the DNA-modified Fe3O4 particles and the DNA template in an incubation solution for co-incubation to obtain the catalyst. Compared with the prior art, this invention solves the problem of the difficulty in achieving precise quantity, accurate position, and consistent spacing of catalysts for CVD growth of CNTs with diameters less than 10 nm in the prior art. This method is based on the programmable characteristics of DNA origami, which allows for precise positioning of Fe3O4 nanoparticles, effectively controlling the spacing, arrangement, and concentration of the catalyst on the substrate, thereby achieving precise control of CVD growth of CNTs using this catalyst.

This invention discloses a chimeric antiviral cross-protective antigen mRNA vaccine, its preparation method, and its application. The vaccine consists of lipid materials encapsulating either a first or second type of mRNA, forming nanolipid nanoparticles containing mRNA fragments. The first type of mRNA is obtained through in vitro transcription using a first DNA template, and the second type of mRNA is obtained through in vitro transcription using a second DNA template. The vaccine of this invention possesses the advantage of multi-antigen comprehensive immunity: each vaccine combines one EEV core surface antigen and one IMV core surface antigen. This chimeric strategy can simultaneously induce highly efficient and specific immunity against both types of viral infection particles, both limiting initial viral entry into cells and effectively clearing free viruses from the circulatory system. It not only stimulates strong humoral immunity but also induces specific cellular immune responses, thereby significantly enhancing the vaccine's broad-spectrum cross-protective efficacy against orthopox, vaccinia virus, and other orthopox viruses.

A preparation method of a super high dose loaded compressed nucleic acid DNA drug delivery platform, according to the disease type, select CpG type, mix the 5' end phosphorylated DNA template containing CpG sequence with the first primer solution, add T4 ligase buffer, mix with ultrapure water to form a circularization system, heat annealing in a PCR instrument; add T4 DNA ligase solution, overnight reaction, after the reaction is completed, inactivate the ligase, obtain the circular DNA template; add phi29 DNA polymerase, 10x phi29 buffer, mixed solution of dATP, dTTP, dCTP, dGTP, Milli-Q to the circular DNA template to form a reaction solution, and the reaction solution is placed in a constant temperature mixing instrument for reaction; then, the second primer and the third primer are added, and constant temperature reaction is carried out to form a DNA drug delivery platform; after the reaction is completed, the polymerase is inactivated, and the reaction is terminated. The application also provides application of the above-mentioned DNA drug delivery platform in preparation of treatment of cancer or inflammatory diseases. The application can super-compress and efficiently load nucleic acid drugs.

FIELD: biotechnology.SUBSTANCE: plasmid DNA matrix pVAX-C3-H1-PolyA and the polynucleotide mRNA vaccine synthesized using the said DNA matrix pVAX-C3-H1-PolyA, encoding the hemagglutinin of the influenza A / H1N1 virus and ensuring its expression and induction of a specific immune response when introduced into the body of mammals, used in immunology, genetic engineering, biotechnology and medicine. A recombinant plasmid DNA matrix pVAX-C3-H1-PolyA was obtained, providing expression of mRNA in mammalian cells, having the nucleotide sequence SEQ ID NO: 1 with a size of 4849 bp, a molecular weight of 3.1 MDa and including a fragment of 1680 bp, having coordinates from 699 to 2378 bp and containing an artificial gene encoding the hemagglutinin protein of the influenza A / Wisconsin / 67 / 2022 (H1N1)pdm09 virus without the transmembrane and cytoplasmic domain with the addition of the trimerizing domain of phage T4 with the initiation codon ATG. An mRNA vaccine was obtained having the nucleotide sequence SEQ ID NO: 2, obtained using a DNA matrix and containing an open reading frame encoding the amino acid sequence SEQ ID NO:3 of the influenza virus hemagglutinin immunogen protein, and ensuring the synthesis and secretion of the said protein in the body of mammals.EFFECT: expansion of the range of more relevant mRNA vaccines against the influenza virus.2 cl, 6 dwg, 3 ex

The application provides a repeated protein expression method and integrated device using anti-degradation circular DNA and circular RNA. The repeated protein expression method comprises a DNA template capable of producing circRNA in a cell-free protein expression system; the DNA template is a double-stranded DNA template with closed ends formed by two single-stranded DNAs; the double-stranded DNA template is connected with a closed linker at two ends of a linear DNA template; the closed linker comprises a 5' end linker and a 3' end linker; the sequence of the non-coding region of the 5' end linker and the sequence of the non-coding region of the 3' end linker can be transcribed into a cleavage site of a circRNA precursor, and a circRNA is formed through a sequence complementary affinity effect. According to the technical scheme of the application, the circRNA can be continuously produced in the cell-free protein expression, the degradation of the DNA fragment is solved, the protein expression efficiency is improved, and the template can be repeatedly used after the hydrogel is covalently bonded with the template, so that the production cost is further reduced.

The application relates to a high-sensitivity and high-efficiency detection method for the quantity of Noctiluca scintillans gametes, which comprises the following steps: filtering and collecting microalgae cells from a water sample to be detected, and extracting a DNA template by adopting a Chelex-100 chelation method; constructing a ddPCR reaction system, wherein the ddPCR reaction system comprises the DNA template, a specific primer pair and a TaqMan probe, the sequence of the specific primer pair is an Ns175F primer shown in SEQ ID NO. 1 and an Ns268R primer shown in SEQ ID NO. 2, and the sequence of the TaqMan probe is shown in SEQ ID NO. 3, the 5' end of the probe is marked with a fluorescent reporter group, and the 3' end is marked with a fluorescent quenching group; performing PCR amplification on the ddPCR reaction system after microdroplet segmentation; reading the fluorescent signal of the PCR amplification product, calculating the initial concentration of the target DNA according to the positive microdroplet proportion, and converting to obtain the concentration of Noctiluca scintillans gametes in the water sample to be detected. The method can solve the problems of low detection sensitivity, complicated operation and inaccurate quantification of low-abundance samples in the prior art.

This invention discloses a CRISPR-Cas12b system-mediated one-step single-tube detection method for *Shewanella algae*, specifically comprising the following steps: (1) mixing the DNA template, MCDA primers, CRISPR-Cas12b / gRNA complex, fluorescent probe, Bst strand displacement DNA polymerase, and 2× DNA polymerase buffer of the sample to be tested to obtain a reaction system; (2) performing MCDA-mediated isothermal amplification and CRISPR-Cas12b-mediated trans-cutting reaction on the reaction system obtained in step (1) at a constant temperature to obtain a mixed MCTOS product; (3) detecting the fluorescence signal or using a lateral chromatography test strip to detect the mixed MCTOS product after the reaction is completed. The detection method of this invention has the advantages of high sensitivity, strong specificity, and simple operation, and is suitable for large-scale application in the detection of *Shewanella algae*.

This invention discloses reporter gene plasmids based on the promoters of the IL-1β and IL-8 genes of the red snapper, their construction methods, and applications, belonging to the field of genetic engineering technology. Using the promoter sequences of the IL-1β or IL-8 genes of the red snapper shown in SEQ ID NO: 1 or SEQ ID NO: 2 as DNA templates, PCR amplification is performed using primers IL1β-promo-KpnI-1F / 1R or IL8-promo-KpnI-1F / 1R to obtain homologous recombination insert fragments A or B. Homologous recombination insert fragments A or B are covalently ligated to a linearized pGL6-TA reporter vector to form circular recombination reporter gene plasmids A or B. The dual reporter gene plasmids provided by this invention are specifically targeted at the red snapper, solving the problem that existing reporter gene plasmids for other fish are ineffective for detecting red snapper due to species differences.