166 results about "Dna template" patented technology

Provided is a circular multimeric tandem RNA sense strand, comprising at least one sense strand sequence and at least one spacer sequence. The circular RNA is derived from an engineered parental DNA template containing all essential sequences, and sequentially comprises a first cyclization element, optionally at least one first restriction enzyme recognition sequence, at least one target sequence, optionally at least one second restriction enzyme recognition sequence, and a second cyclization element. The circular multimeric tandem RNA sense strand can bind and deliver a plurality of antisense strand RNA, increasing the binding of the sense strand and the antisense strand while utilizing the stability advantage of circular RNA.

The invention provides annular multi-tandem PCSK9 siRNA, which comprises at least one positive-sense strand sequence and at least one spacer sequence. The sense strand of the cyclic siRNA is derived from an engineered parent DNA template containing all essential sequences, comprising in the following order a first cyclization element, optionally at least one first restriction enzyme recognition sequence, at least one target sequence, optionally at least one second restriction enzyme recognition sequence, and a second cyclization element. The annular multi-tandem RNA positive-sense strand can combine and deliver a plurality of antisense strand RNAs, and the combination of the positive-sense strand and the antisense strand is increased while the stability advantage of the annular RNA is utilized.

Provided is a circular concatemeric PCSK9 siRNA, comprising at least one sense strand sequence and at least one spacer sequence. The sense strand of the circular siRNA is derived from an engineered parental DNA template containing all essential sequences, which comprises, in the following order: a first cyclization element, optionally at least one first restriction enzyme recognition sequence, at least one target sequence, optionally at least one second restriction enzyme recognition sequence, and a second cyclization element. The sense strand of the circular concatemeric RNA can bind and deliver a plurality of antisense strand RNAs, enhancing the binding between the sense and antisense strands while leveraging the stability advantage of the circular RNA.

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 relates to the field of in vitro transcription, and particularly to a plasmid backbone capable of improving the stability of polyadenylate tails. The present invention provides an expression cassette, which sequentially includes: the rop gene, the replicon ori, and the resistance gene; the rop gene, the replicon ori, and the resistance gene are connected by a linking fragment. The present invention has established a low-copy pmRVacSL and a medium-copy pmRVacM plasmid vector system through artificial modification. Since pmRVacSL is a low-copy vector, the yield of plasmid DNA is relatively low. The pmRVacM plasmid backbone solves the problem of relatively low plasmid DNA yield of pmRVacSL. This vector system can not only maintain the stable replication of polyA tail sequences of more than 150 bp, but also quickly add genes of interest and polyadenylate (polyA) sequences, and efficiently and rapidly obtain the DNA template for in vitro transcription of the target mRNA.

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 belongs to the field of plant molecular genetics, and relates to a spirodela polymorphic microsatellite molecular marker primer and application thereof. The kit comprises 10 pairs of microsatellite primers which form 5 groups of duplex PCR systems, and the kit can be applied to population genetic diversity analysis of spirodela polyrhiza. The population genetic diversity analysis comprises the following steps: collecting a sample of spirodela polyrhiza to be identified; extracting spirodela polyrhiza genome DNA from the spirodela polyrhiza sample; taking the extracted DNA as a template, and carrying out PCR (Polymerase Chain Reaction) amplification on the DNA template by using the screened 10 pairs of microsatellite primers; carrying out electrophoresis detection on a PCR product, and then carrying out silver staining; the silver staining result is digitally analyzed, the UPGMA clustering analysis chart is drawn by genetic analysis software, the clustering analysis chart of the spirodela polyrhiza is identified according to the clustering analysis result, and the method has huge popularization and utilization value.

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 belongs to the technical field of microbiological detection, and particularly relates to a real-time fluorescent quantitative PCR (polymerase chain reaction) primer, a probe and a kit for detecting a special drug-resistant pedigree of candida albicans. The nucleotide sequence of the specific primer F is as shown in SEQ ID NO. 1; the nucleotide sequence of the specific primer R is as shown in SEQ ID NO. 2; the nucleotide sequence of a probe matched with the primer for use is as shown in SEQ ID No.3. The kit prepared by the invention ensures high specificity and high sensitivity in a PCR amplification process, effectively distinguishes drug-resistant mutant strains from sensitive strains, keeps efficient detection capability in a low-copy-number DNA template, reduces non-specific amplification and background signal interference, and improves the reliability and accuracy of a detection result.

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 relates to the technical field of gene engineering, in particular to a method for rapidly and economically carrying out gene function analysis on lepidoptera insects and application. The method comprises the following steps: designing four different sgRNAs by aiming at an exon region of a target gene, and constructing a sgRNAs synthetic primer mixing pool; 4 different sgRNA-DNA templates are synthesized at the same time, 4 different sgRNAs are further synthesized at the same time, and a sgRNAs mixed pool is formed; the sgRNAs-Cas9 protein and Cas9 protein are prepared into an sgRNAs-Cas9 compound, and the sgRNAs-Cas9 compound is microinjected into newly laid eggs; after the injected eggs develop into larvae, genome DNA is extracted, sgRNAs targeted areas are amplified, the mutation rate of target genes is determined through T7E1 enzyme digestion, and phenotype analysis is conducted on the larvae. According to the method, the mutation rate of the target gene is increased through redundant targeting of the four sgRNAs, so that functional analysis of the target gene is quickly realized in the G0 generation, and the research period of the gene function of the lepidoptera insects is greatly shortened. Besides, four different sgRNAs are synthesized at the same time by constructing a primer mixing pool of a plurality of sgRNAs, so that the economic cost for synthesizing the sgRNAs is greatly reduced.

The invention relates to a kit for detecting Tau-181 protein and application, and relates to the technical field of biological detection.The kit comprises a first antibody coupled with a first component of DNAzyme with catalytic activity, a second antibody coupled with a second component of DNAzyme with catalytic activity, phi29DNA polymerase, dNTPs, an annular DNA template, an initiation primer, a fluorescent beacon probe and a reaction buffer solution; the Tau-181 protein detection technology based on double-antibody sandwich coupling DNAzyme is used for quantitatively detecting the content of the Tau-181 protein in a biological sample in vitro, and can be applied to the fields of neuroscience research and clinical diagnosis, especially in the processes of early diagnosis, disease monitoring and drug research and development of neurodegenerative diseases such as Alzheimer's disease. And the method has important significance in the aspects of detection and analysis of a disease-related biomarker Tau-181 protein and the like.

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 discloses a primer, a kit and an identification method for specifically identifying sheepskin, and belongs to the technical field of leather identification. The specific primer comprises a KLHL17 forward primer as shown in SEQ ID NO. 1 and a KLHL17 reverse primer as shown in SEQ ID NO. 2. The invention also discloses a kit for detecting the specific primer. The identification method specifically comprises the following steps: 1) extracting DNA of a cortex sample to be detected to obtain a total DNA template; (2) carrying out PCR (Polymerase Chain Reaction) amplification reaction by adopting a KLHL17 forward primer, a KLHL17 reverse primer and the total DNA template to obtain an amplified DNA fragment; and (3) detecting and amplifying the DNA fragment by adopting agarose gel electrophoresis, and judging that the sample is sheep skin if the size of a PCR (Polymerase Chain Reaction) amplified band is 106bp. The method disclosed by the invention has the characteristics of rapidness, simplicity, convenience, accuracy, wide application range and low requirements on equipment, and can be used for remarkably improving the identification efficiency and reducing the detection cost. In addition, the wide application of the method is of great significance to standardize the leather market order and improve the accuracy of leather product traceability.

The present invention provides a reagent for detecting the DNA barcode of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. and a fluorescence detection method for Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. The reagent of the present invention includes a specifically upstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. modified with AuNPs and a specifically downstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. modified with a fluorescent label. The sequences of the specifically upstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. and the specifically downstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. modified with a fluorescent label are respectively: 5’-SH-(CH2)6-ATTGACCACGCGAACTTGTGA-3’; 5’-fluorescent label-ACCGTCAACAAACCTGATTGCT-3’. Detection method: Mix the specifically upstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. modified with AuNPs and the specifically downstream primer of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag. modified with a fluorescent label with a DNA template solution of the to-be-detected Asarum species for PCR reaction amplification, and detect the fluorescence spectral value of the obtained product to achieve qualitative or quantitative detection of Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.) Kitag.

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 present invention provides a circular DNA template precursor for detecting Clostridium difficile, a preparation method thereof and an application thereof. The circular DNA template precursor includes a three-dimensional self-assembly reaction using a reaction system composed of H1 and H2 hairpin probes and a trigger strand Trigger. The three-dimensional self-assembly reaction includes Trigger triggering the H1 hairpin structure to form a T / H1 intermediate, the H1 hairpin structure being unfolded and binding to H2 to produce an H1 / H2 structure, and the DNA template of H2 being cyclized to obtain the circular DNA template precursor. When Clostridium difficile toxin is present, the trigger strand Trigger can be released again, and can continuously catalyze the hairpin structure of the H1 DNA hairpin probe without being consumed. By adopting the technical scheme of the present invention, signal cascade three-stage amplification is realized through three-dimensional self-assembly-amplification reaction and silver-enhanced gold nanoparticles, so as to realize highly sensitive and highly specific detection of Clostridium difficile infection.

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.

The present invention relates to a bioreactor for RNA in vitro transcription, a method for RNA in vitro transcription, a module for transcribing DNA into RNA and an automated apparatus for RNA manufacturing. Further, the use of a bioreactor for RNA in vitro transcription as described herein is part of the present invention. The present invention relates to an RNA in vitro transcription reactor designed to be operable in an automated manner under GMP-compliant conditions. In particular, said RNA in vitro transcription reactor allows repetitive use of DNA template for various RNA in vitro transcription reactions. Further, the invention relates to an apparatus for RNA manufacturing comprising (a) a module for template DNA synthesis, (b) a module for transcribing DNA into RNA comprising said RNA in vitro transcription reactor, and, optionally, (c) a module for RNA formulation.

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 present invention discloses a capillary monolithic column modified with a deoxyribozyme nanoflower, a preparation method thereof, and an application thereof in separating chiral molecules, belonging to the field of pharmaceutical analysis. The present invention synthesizes a deoxyribozyme-based nanoflower chiral selector, the nucleotide sequence of the single-stranded DNA template of the deoxyribozyme being shown in SEQ ID NO.1, and the synthesized deoxyribozyme nanoflower having good pH and temperature stability. The prepared deoxyribozyme nanoflower is bonded to a graphene oxide-functionalized capillary silica monolithic column, and chiral molecules are separated under electrophoretic conditions, achieving baseline separation of 2'-deoxyadenosine, atenolol, propranolol, tyrosine, and nafopam enantiomers.

The invention provides a library building method and application of a genomic library and a methylated library. The construction method comprises the following steps: carrying out linker connection on a DNA template subjected to terminal repair, and carrying out PCR (Polymerase Chain Reaction) amplification on a connection product to obtain a mixture of a copy chain and an original chain; separating the mixture of the copy chain and the original chain, and respectively performing genome library construction and methylation library construction by using the obtained copy chain group and the original chain group to obtain a genome library and a methylation library; wherein the 3'end of the linker sequence connected with the 3 'end of the DNA template is modified by biotin. According to the present invention, the 3'end of the DNA template is connected with the biotin-modified linker, such that the effective separation of the copy chain and the original chain is achieved, the same DNA template can be adopted to respectively construct the genome library and the methylation library, and the information of the genome and the methylation genome can be comprehensively and accurately obtained.

The invention discloses a cdLAMP-based detection kit for simultaneously detecting staphylococcus aureus and salmonella and a detection method thereof, and relates to the field of biological detection.The detection kit comprises a silicon-based nano porous chip, a primer probe combination, 20 mM Tris-HCl, 10 mM KCl, 8 mM MgSO4, 10 mM (NH4) 2SO4, 0.05% Tween-20, a 2.5 mu L DNA template, 1.4 mM dNTP, 0.16 U / mu L Bst DNA polymerase and DEPC; the primer probe combination comprises a primer probe and a primer probe, wherein the primer probe is designed according to a nuc gene of staphylococcus aureus: GenBank registration number EF529607.1, and the primer probe is designed according to a fimY gene of salmonella: GenBank registration number JQ665438.1; according to the invention, dual detection is realized through a chip and a two-color fluorescence channel carried by an imaging analyzer, and compared with the existing detection method, the method has higher sensitivity and accuracy.

The invention discloses a two-color fluorescent aptamer sensor for detecting kanamycin and aflatoxin B1 in feed by using two-color fluorescent silver nanoclusters, and belongs to the field of food safety detection. According to the invention, different DNA templates are utilized to synthesize the double-color silver nano-cluster as a fluorescent probe, and the double-color silver nano-cluster has two different fluorescent colors of 530 nm and 630 nm. And a sequence rich in guanine is used as a capture probe, so that an amplification effect on signals of two kinds of fluorescence is achieved. When KAN or AFB1 exists, a corresponding target object is selectively combined with an aptamer fragment to form an aptamer-target object-aptamer sandwich structure, and the fluorescence intensity of the silver nano-cluster is induced to be enhanced through G-rich sequence and base pairing. The'lightening 'type double-color fluorescent aptamer sensor shows excellent detection performance, the KAN detection limit reaches 0.86 nM, the AFB1 detection limit reaches 0.71 nM, the'lightening' type double-color fluorescent aptamer sensor can be used for detecting kanamycin and aflatoxin B1 in a feed sample, and the reliability and application potential of the'lightening 'type double-color fluorescent aptamer sensor in multiple detection of antibiotics and toxins in the feed sample are highlighted.

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.

A method for transcribing a DNA template into RNA in vitro includes providing a mixture comprising a buffer material, ribonucleoside triphosphate (NTP), one or more magnesium salts at a concentration of from about 2 mM to about 60 mM, the DNA template, and a recombinant RNA polymerase, and incubating the reaction mixture at from about 25 DEG C to about 40 DEG C for from about 1 hour to about 12 hours, thereby producing the RNA. In one embodiment, a method for in vitro transcription includes providing a DNA template and a cap analog that binds-1 and / or + 1 nucleotides of a promoter for in vitro transcription, thereby producing mRNA of more complete length, allowing for more flexible selection of a first mRNA base, and providing a + 2 position open to a custom sequence.