88 results about "Dna template" patented technology

The invention relates to the technical field of phytopathogen detection, and particularly discloses a primer, a kit and a method for detecting crop root rot fusarium solani and application, the primer comprises a specific RPA primer RPA-F and a specific RPA primer RPA-R; the specific RPA primer RPA-F is as shown in SEQ ID NO. 1, and the specific RPA primer RPA-R is as shown in SEQ ID NO. 2; the kit further comprises a guide RNA primer crRNA, and the guide RNA primer crRNA is as shown in SEQ ID NO. 3. The kit comprises a specific RPA primer RPA-F, a specific RPA primer RPA-R, a Cas12 protein, a T7 transcriptase, a crRNA DNA template, a DNA polymerase and a Cas12 / 13 nucleic acid detection test strip. The kit has the characteristics of good sensitivity, relatively good specificity and enough reliability.

The present invention provides a process for the production of a closed linear DNA comprising the steps of (a) providing a DNA template comprising a DNA sequence of interest; (b) amplifying DNA from the DNA template of step (a) wherein the amplification is primed with a primase / polymerase enzyme; (c) generating a closed linear DNA with the amplified DNA produced in step (b); and (d) purifying the closed linear DNA produced in step (c). The invention also provides a closed linear DNA obtainable according to the process of the invention, a pharmaceutical composition comprising a therapeutically effective amount of the closed linear DNA of the invention, and a concatameric DNA comprising repeats of a DNA sequence of interest.

The invention provides a microsatellite genotyping method, and belongs to the technical field of genotyping. According to the microsatellite genotyping method, a bar code primer is introduced into a microsatellite primer, and the bar code primer comprises a bar code and a universal primer. The universal primer can be used for increasing PCR products obtained by the microsatellite primer so as to supplement a target product obtained by the microsatellite primer, the problem of amplification of a low-quality DNA template is solved, the library building success rate is remarkably increased to 90-100% from 60-70%, the library building and sequencing times are remarkably reduced, the sample number of each time of library building and sequencing is increased, and the library building and sequencing efficiency is improved. The method has the remarkable advantages of low cost and high accuracy.

The invention provides an ISSR-PCR reaction system for identifying kiwi fruit varieties with different colors and a method and application of the ISSR-PCR reaction system, and belongs to the technical field of molecular markers. The ISSR-PCR reaction system for identifying the kiwi fruit varieties with different colors is as follows: the DNA template is 70ng / mu L, the primer is 1.0 mu mol / L, the Mg < 2 + > is 2mM, the mix is 10mu L, and the remaining ddH2O is prepared into 20 mu L of the system. The amplification procedure is as follows: the pre-denaturation temperature is 94 DEG C, and the time is 5 minutes; the denaturation temperature is 94 DEG C for 30 seconds; the annealing temperature is set according to different primer conditions, and the time is 50s; the extension temperature is 72 DEG C, and the extension time is 1 min; circulation is performed for 35 times; and then extending at 72 DEG C for 7 minutes, and storing at 10 DEG C. The system is used for amplifying 42 kiwi fruit germplasm resources with different colors, the amplification result shows that the established ISSR-PCR reaction system is good in repeatability and high in stability, and the established ISSR molecular fingerprint spectrum can be used for variety identification.

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.

The invention provides an in-vitro transcription method based on RNA polymerase and a kit, which are suitable for the fields of mRNA synthesis, gene research and biological pharmacy. According to the method, the problems of low efficiency, poor stability and the like of traditional in-vitro transcription are solved through an optimization technology. The method comprises the following specific steps: providing a linear DNA template containing a T7 promoter; according to the method, site-directed mutagenesis or fusion protein modified high-activity T7RNA polymerase is combined with chemically modified NTPs and a spermine-BSA compound stabilizer to react under the conditions that the temperature is 37-42 DEG C and the pH is 7.5-8.5, and formation of double-stranded RNA is promoted through dynamic temperature control. A reaction system contains Mg < 2 + > / Mn < 2 + > and DTT, and enzyme activity and product integrity are optimized. The kit comprises an improved reagent and a standardized process, the transcription efficiency is improved by 2-5 times, the product yield is greater than or equal to 80%, and the activity is not lost after the kit is stored at 4 DEG C for 1 month. The method has the technical advantages that NTPs degradation resistance is achieved, template preparation is simplified, universality is high, the method is suitable for large-scale production of mRNA vaccines, gene therapy and the like, the production cost is remarkably reduced, and wide application of the in-vitro transcription technology is promoted.

The invention relates to the field of DNA (Deoxyribose Nucleic Acid) sequencing, in particular to a pathogenic microorganism high-throughput sequencing detection method and a pathogenic microorganism high-throughput sequencing detection system based on multiple PCR (Polymerase Chain Reaction) detection. The method comprises the following steps: quantitatively adding an internal reference nucleic acid marker into a first microorganism sample to be detected to generate a standardized DNA template library; performing multiple PCR amplification and quality control judgment and evaluation on the standardized DNA template library to generate a PCR quality control result table; constructing a sequencing library based on the PCR quality control result table to generate sequencing library original data; performing host reference genome comparison and removal on the original data of the sequencing library, and extracting a data set to be compared; and performing pathogenic species comparative analysis according to the to-be-compared data set, and outputting a final pathogenic microorganism detection list. The invention realizes rapid and accurate detection of pathogenic microorganisms.

The invention provides a plasmid and application thereof. The plasmid comprises a recognition site of a first endonuclease, a target gene and a recognition site of a second endonuclease from a 5 '-3' direction. According to the invention, the plasmid is amplified by using a rolling circle amplification method, the rolling circle amplification product is cleaved by using the first endonuclease and the second endonuclease, the enzyme cleaved product is digested by using exonuclease, and the product is further purified by using oligonucleotide. The obtained product can be used for in vitro transcription or directly applied to eukaryotic expression. According to the scheme provided by the invention, a simple, high-yield, economic, rapid and amplifiable preparation process of the full chemical synthesis DNA template for escherichia coli fermentation is established.

Provided are a circular functional nucleic acid, a preparation method therefor and a use thereof. The method involves combining a functional nucleic acid with a circular nucleic acid, using a method combining DNA template-directed assembly with enzymatic conjugation to first prepare a single-stranded circular DNA, synthesize a corresponding outer short strand, and conjugate a functional nucleic acid drug onto the outer short strand, and optimizing and screening reaction conditions, thereby preparing a circular functional nucleic acid. The circular functional nucleic acid has the characteristics of adjustable size, adjustable valence state (multivalent or polyvalent), high stability, and the capability for chemical conjugation with drugs or drug incorporation, etc.; a novel circular multivalent functional nucleic acid technology is created, which significantly enhances the targeting ability and stability of nucleic acid drugs; thus, a nucleic acid drug development platform o-FLARE having efficient targeting, immune agonism, and protein degradation functions can be established.

A method and composition for preparing a DNA library for replicating a target nucleic acid sequence are provided. A target DNA template containing the target sequence is circularized via a terminal adapter to form a circular construct, which is extended bidirectionally by polymerase-mediated elongation initiated at a nick site on the terminal adapter. Following polymerase-mediated elongation, a diploid DNA template is formed containing two copies of the target DNA template (and thus two copies of the target sequence). Each strand of the diploid DNA template contains a parent polynucleotide chain ligated to a newly synthesized daughter strand copy of the parent polynucleotide chain. Predetermined sequences, such as primer sequences, unique molecular identifiers, and sequence indices, can be included in the diploid DNA template. Sequencing of the diploid DNA template can reveal genetic / epigenetic information related to the target sequence. A method for constructing asymmetric and multiploid DNA template constructs is also provided.

The application discloses a primer, a kit and an identification method for identifying a leather cultural relic species, and belongs to the field of leather cultural relic molecular archaeology. The application provides an upstream primer cytbF shown as SEQ ID NO. 1 and a downstream primer cytbR shown as SEQ ID NO. 2. The specific method is as follows: (1) extracting DNA of a leather sample to be detected to obtain a total DNA template; (2) performing PCR amplification reaction on the total DNA template by using the upstream primer cytbF and the downstream primer cytbR to obtain an amplification product; (3) performing bidirectional sequencing or second-generation sequencing on the amplification product, and performing BLAST similarity comparison on the sequencing result to determine the species of the leather sample to be detected. The primer can be used not only for amplification of single leather sample DNA, but also for mixed leather samples, and in addition, can be used for identification of leather samples damaged by acid and alkali in the environment. The identification method provided by the application is rapid, simple and accurate, and has important significance in the field of cultural relic identification, protection and restoration.

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.

The invention belongs to the technical field of biology, and particularly relates to a DNA polymerase mutant for improving amplification efficiency of a high GC sample and application of the DNA polymerase mutant. According to the invention, DNA polymerase mutants KODG and KODC are obtained through site-specific mutagenesis, and on the basis of retaining the capability of amplifying a DNA template with conventional GC content, the capacity of amplifying a DNA template with high GC content (gt; compared with the prior art, the method has the advantages that the amplification efficiency of a DNA template (3kb, 60%) is improved, effective amplification of difficult-to-amplify fragments with 3kb, 95% GC content and the like is successfully realized, and the technical bottleneck of amplification failure caused by high thermal stability and difficulty in complete melting of the existing polymerase in a high-GC region is overcome, so that the application range of the DNA polymerase in complex template amplification is expanded, and the application prospect is broad. And a more reliable enzymology tool is provided for related detection and identification of high-GC-content samples.

The invention discloses a QPCR (Quantitative Polymerase Chain Reaction)-based DNA (Deoxyribose Nucleic Acid) methylation rate detection method, which comprises the following steps of: treating a DNA sample to be detected by bisulfite, dividing the DNA sample into a first sample and a second sample which are parallel to each other, and performing substrate amplification on a methylated DNA template and a non-methylated DNA template in the samples through PCR amplification by adopting the same amplification primer under the same condition; then respectively depleting dATP in a sample A reaction system and dGTP in a sample B reaction system through a template and a primer with thymine deoxyribonucleotide repetitive sequences or a template and a primer with cytosine deoxyribonucleotide repetitive sequences, and controlling independent detection of methylation and non-methylation templates through a d3TP system lacking dATP or dGTP; and finally, directly analyzing the fluorescence signal of the methylation template and the fluorescence signal of the non-methylation template, and calculating the methylation rate of the DNA sample to be detected through a methylation rate calculation function.

This invention discloses a CRISPR / Cas-regulated DNA silver nanocluster sensor and its method and application for detecting adulteration in meat, belonging to the field of food safety testing technology. This DNA-templated silver nanocluster sensor introduces a novel tagless fluorescent probe, DNA-AgNCs, as a substrate for CRISPR / Cas12a. After the CRISPR / Cas12a-crRNA complex specifically recognizes the target nucleic acid, its trans-cleavage activity is activated, leading to the cleavage of the DNA template and a significant decrease in fluorescence signal. Detecting adulteration in meat using this DNA-templated silver nanocluster sensor can cleverly convert the concentration of adulteration into a quantifiable fluorescence signal output, achieving quantitative detection of adulterants. It exhibits high sensitivity and specificity, providing a universal and sensitive detection method for meat adulteration.

The invention discloses a hot-start high-specificity LAMP (loop-mediated isothermal amplification) isothermal nucleic acid rapid detection method which comprises the following operation steps: step 1, preparing three pairs of LAMP amplification primers, the 3'ends of one pair of primers F3 and B3 being one of ddC / ddG / ddT / ddA or other closed groups, and each of the primers F3 and B3 internally containing an RNA (ribonucleic acid) basic group; 2, establishing an LAMP isothermal nucleic acid amplification reaction system by using three pairs of primers; 3, in the process of hot start of the high-specificity LAMP amplification reaction, after the primers F3 and B3 are paired with the template DNA, cutting off the DNA at the RNA base by thermal stability endonuclease HII to generate a double-dumbbell initiator (a product 6 in the figure 1), thereby starting the LAMP continuous amplification reaction; if the primers F3 and B3 cannot be specifically paired with the DNA template, the primers F3 and B3 cannot be cut by thermal stability endonuclease HII, and a double-dumbbell type initiator cannot be generated, so that the LAMP continuous amplification reaction cannot be started.

In one aspect, the disclosure relates to a system for site-specific modification of a double­stranded target DNA sequence, the system including at least: a fusion protein comprising an RNA-binding nickase protein and a DNA-dependent DNA polymerase, and a chimeric guide nucleic acid sequence comprising a guide RNA sequence that interacts with the RNA-binding nickase protein and a single-stranded DNA template, wherein the single-stranded DNA template comprises a modified sequence for insertion into a second strand of the double-stranded target DNA sequence and a primer binding site. Also disclosed is a method for site-specific modification of a double-stranded target DNA sequence in a cell. In another aspect, the present disclosure is directed to a fusion protein including an RNA-binding nickase protein and a DNA-dependent DNA polymerase, wherein one or both of the nicakse protein and the DNA polymerase can include one or more mutations to optimize function and prevent off-target effects.

The invention discloses a hairpin type DNA-copper cluster probe for rapid ratio fluorescence detection of Hg < 2 + > and a preparation method, the hairpin type DNA-copper cluster probe contains a hairpin type DNA template (hpDNA) capable of emitting strong red fluorescence and weak blue fluorescence at the same time, and the sequence of the hairpin type DNA template is 5 '-TTTTTTTTTTTTTTTTTTTTT TTTTCGCGCGATATATCGCGCG-3'. The method comprises the following steps: preparing a single-stranded DNA aqueous solution by using a 3-morpholine propanesulfonic acid buffer solution, heating the single-stranded DNA aqueous solution in a constant-temperature water bath kettle, cooling to room temperature, and refrigerating and storing in a refrigerator to form an hpDNA template solution; adding a copper sulfate solution into the hpDNA template solution, and incubating at constant temperature to obtain a mixed solution; and adding a sodium ascorbate (SA) solution into the mixed solution, reacting at a constant temperature, and reducing Cu < 2 + > coordinated on the hpDNA template by sodium ascorbate to obtain the hairpin type DNA-copper cluster probe for rapid ratio fluorescence detection of Hg < 2 + >. The hairpin DNA-copper cluster (hpDNA-CuNCs) probe can be used for rapid ratio fluorescence detection of Hg < 2 + >. The hpDNA-CuNCs probe has excellent selectivity on Hg < 2 + >, is used for rapid and sensitive detection of trace Hg < 2 + > in an actual water body sample, and has an accurate detection result.

The present invention relates to the field of in-vitro transcription, in particular to a plasmid backbone capable of improving the stability of the polyadenylic acid tail. Provided is an expression cassette, sequentially comprising: a rop gene, a replicon ori, and a resistance gene, wherein the rop gene, the replicon ori, and the resistance gene are connected by means of linker fragments. Low-copy pmRVacSL and medium-copy pmRVacM plasmid vector systems are established by means of artificial modification. Since pmRVacSL is a low-copy vector, the plasmid DNA yield is relatively low. The pmRVacM plasmid backbone addresses the problem of low plasmid DNA yield of pmRVacSL. The vector system not only enables stable replication of polyA tail sequences of 150 bp or more, but also allows for rapid addition of genes of interest and polyadenylic acid (polyA) sequences, efficiently and rapidly obtaining the target DNA template for mRNA in vitro transcription.

The invention provides a method, a system and a non-volatile computer readable medium for efficiently designing a large-scale multiplex PCR (Polymerase Chain Reaction) primer group through a DIMPLE (Degenerate Incomplete Multiplex Primer List Extension) technology. The DIMPLE has shorter running time, and can be expanded to large-scale multiple application. The technology is suitable for PCR primer design of a standard DNA template and a post-transformation DNA template containing degenerate nucleotide. The DIMPLE can design a plurality of primers for each target DNA template at the same time, so that experimental optimization is facilitated, and nested PCR is supported. Finally, the DIMPLE can avoid the use of an improper template, thereby reducing the experiment failure rate.

The invention discloses a preparation method of threose nucleic acid based on a self-pairing primer template. The method comprises the following operation steps: synthesizing a hairpin-shaped DNA template modified by biotin and hypoxanthine, and carrying out annealing treatment to realize template self-pairing; preparing a TNA sequence in a reaction system compatible with TNA extension and TNA cutting by taking the self-pairing DNA hairpin as a template; the TNA product and the template are separated through the magnetic beads, and the DNA template combined with the magnetic beads is recycled, so that the TNA product is recycled in multiple rounds of reactions, and the target TNA product is prepared in batches; the molecular weight of the product is detected through electrospray ionization mass spectrometry, and the prepared TNA is determined as an expected product. The invention provides a method for rapidly synthesizing a large amount of TNA sequences, the preparation period of TNA is shortened, and technical support is provided for application of TNA in the fields of biotechnology and biomedicine.

A method for inserting a polynucleotide exogenous transgene sequence at a predetermined endogenous genetic locus in the genome of a host cell, the method comprising: (i) a donor DNA template comprising a polynucleotide insertion fragment; 5'homologous arms; and a 3'homologous arm. In some embodiments, the 5'homologous arm and the 3 'homologous arm are complementary to DNA in the target region; and (ii) a ribonucleoprotein complex (RNP) comprising (1) a Cas nuclease and at least one small guide RNA (sgRNA) complementary to at least one selected nucleic acid sequence within the predetermined genetic locus in the host cell genome.

A method for amplifying a DNA template, comprising: a) providing a composition comprising a DNA template, an RNA polymerase, a DNA polymerase, and a deoxyribonucleotide, wherein said composition does not comprise a ribonucleotide; and b) incubating said composition in a suitable condition to allow amplification of said DNA template. Also provided is a method for priming DNA amplification from a DNA template, comprising: incubating a DNA template, an RNA polymerase and a deoxyribonucleotide in a suitable condition to allow the initiation of DNA amplification; wherein said method does not comprise adding a ribonucleotide.

The identification of mutations that are present in a small fraction of DNA templates is essential for progress in several areas of biomedical research. Though massively parallel sequencing instruments are in principle well-suited to this task, the error rates in such instruments are generally too high to allow confident identification of rare variants. We here describe an approach that can substantially increase the sensitivity of massively parallel sequencing instruments for this purpose. One example of this approach, called “Safe-SeqS” for (Safe-Sequencing System) includes (i) assignment of a unique identifier (UID) to each template molecule; (ii) amplification of each uniquely tagged template molecule to create UID-families; and (iii) redundant sequencing of the amplification products. PCR fragments with the same UID are truly mutant (“super-mutants”) if ≥95% of them contain the identical mutation. We illustrate the utility of this approach for determining the fidelity of a polymerase, the accuracy of oligonucleotides synthesized in vitro, and the prevalence of mutations in the nuclear and mitochondrial genomes of normal cells.

Provided herein is a DNA-based nanostructure at least a first plurality of virus-binding moieties, wherein each virus-binding moiety of the at least first plurality of virus-binding moiety is neuraminic acid or a derivative thereof. Also provided herein is a self-assembling DNA-based building block formed by a single-stranded DNA template strand and a set of oligonucleotides complementary to said single-stranded DNA template, wherein: each of the oligonucleotides is either complementary to one contiguous DNA sequence stretch or to at least two non-contiguous DNA sequence stretches on said single-stranded DNA template; and the self-assembling DNA-based building block comprises at least one first virus-binding moiety, wherein the first virus-binding moiety is neuraminic acid or a derivative thereof. Also provided herein is a DNA-based nanostructure wherein the DNA-based nanostructure comprises at least five self-assembling DNA-based building blocks; each of the self-assembling DNA-based building blocks is formed by a single-stranded DNA template strand and a set of oligonucleotides that are at least partially complementary to said single-stranded DNA template; and wherein each of the oligonucleotides is either complementary to one contiguous DNA sequence stretch or to at least two non-contiguous DNA sequence stretches on said single-stranded DNA template. Also provided herein are associated methods, uses, compositions, and kits.

The invention belongs to the technical field of biology, and particularly relates to a DNA polymerase mutant for improving long fragment amplification efficiency and application of the DNA polymerase mutant. According to the invention, the DNA polymerase mutants KODL and KODW with enhanced continuous synthesis capability are successfully constructed by carrying out combined mutation of specific amino acid sites on the KOD DNA polymerase, the amplification efficiency of the mutants on a long-fragment DNA template (such as 30kb) is remarkably improved on the basis of retaining the amplification efficiency on fragments with conventional lengths, and the application of the mutants to the preparation of the long-fragment DNA template is facilitated. The technical bottleneck of the existing variable temperature amplification enzyme in long fragment amplification is broken through, and an efficient and reliable enzymology tool is provided for molecular biology application of long fragment gene cloning, whole genome amplification, complex structure analysis and the like.

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.

Described herein is a DNA template plasmid for generating genome modified immune cells including a plasmid backbone, a first insert, and a second insert. The first insert includes a transgene, wherein the transgene is flanked by left and right homology arms that are complementary to sequences on both sides of a cleavage site in a target expressed gene in an unmodified immune cell. The second insert includes a cleavage target including a protospacer sequence defining the cleavage site in the target expressed gene, and a protoadjacent motif sequence (PAM) for recognition by a Cas9 ribonucleoprotein complex (Cas9-RNP). The Cas9-RNP includes a Cas9 polypeptide and a single guide RNA (sgRNA) comprising a sequence complementary to the protospacer sequence. The Cas9-RNP binds the second insert and linearizes the DNA template plasmid by Cas9-RNP-directed cleavage at the cleavage site. Also included are RNP complexes, methods of genome modifying immune cells, and treatment methods.

The application belongs to the technical field of biological medicine, and provides a DNA programmed virus neutralizing multivalent aptamer medicament and a preparation method thereof.The medicament comprises a circular DNA as a template, and a DNA multivalent neutralizing aptamer particle is synthesized through a rolling circle amplification reaction, wherein the DNA template can be designed to be directed against a plurality of virus antigen epitope neutralizing aptamers.The DNA programmed virus neutralizing multivalent aptamer medicament provided by the application is simple to operate, raw materials are easy to obtain, has good biocompatibility, and has a wide application range, can effectively inhibit the invasion of virus particles into the cell interior, can capture and close the virus particles, thereby inhibiting the infection of the virus particles on the host cells, and can play a safe and effective virus neutralizing role and block the transmission and infection of the virus.

The invention belongs to the technical field of virus detection, and discloses a reaction solution for DNA virus and RNA virus detection, the reaction solution is a reaction solution containing acetate, acetate salt is introduced as an ionic reaction system, the specificity of a one-step method RT-PCR amplification system is greatly improved, and the specificity of the one-step method RT-PCR amplification system is greatly improved. The amplification inhibition phenomenon of the residual reverse transcriptase activity on a subsequent DNA template is greatly reduced, so that the kit has a good effect on DNA detection while being capable of detecting the RNA virus.