7 results about "Nucleotide synthesis" patented technology

This invention provides a high-throughput method for constructing dual sgRNA libraries and its applications. The method utilizes high-throughput microarray synthesis technology to prepare a set of DNA fragments containing multiple dual sgRNA expression cassettes in a single step. After amplification, these fragments are assembled with a target vector containing a first promoter and a second gRNA backbone sequence in a first round of directed assembly to obtain a preliminary recombinant plasmid set. Then, linearized enzyme digestion and homologous recombination technology are used to insert a fragment containing a transcription termination sequence and a complete second promoter to complete the construction of the dual sgRNA expression unit. Finally, transformation and amplification yield the dual sgRNA plasmid library. This invention avoids the high error rate and high cost of long-chain oligonucleotide synthesis by utilizing microarray synthesis and simplifies the operation process through two rounds of directed assembly, significantly improving the throughput, fidelity, and efficiency of library construction. It is applicable to the construction of genome-wide dual sgRNA libraries, providing an efficient and reliable technical platform for high-throughput gene function screening, drug target discovery, and gene interaction research based on CRISPR.

Described herein are aptamers for temperature-dependent reversible inhibition of thermostable polymerase activity in order to improve sensitivity and specificity of various reactions and assays involving hot start polynucleotide synthesis. Methods for use of the aptamers and related compositions and kits are also provided.

The application provides a promoter capable of improving the transcription level of a formylglycine-generating enzyme gene and application, the promoter is obtained by mutating the sequence shown in SEQ ID NO:1, and five promoters capable of enhancing the transcription level of a formylglycine-generating enzyme gene and capable of improving the expression of an alpha-amylase amyE gene from Bacillus subtilis subsp. subtilis str. 168 in coryneform bacteria are obtained.

The present disclosure pertains to methods of synthesizing a polynucleotide by associating a polynucleotide scaffold with a plurality of adaptor oligonucleotides and a plurality of fragment oligonucleotides to form a mixture. Each of the adaptor oligonucleotides includes subsequences that become hybridized with the polynucleotide scaffold and other subsequences that become hybridized with subsequences of the fragment oligonucleotides to form adaptor oligonucleotide: fragment oligonucleotide complexes. A ligase is then added to the mixture to ligate the adjacent fragment oligonucleotides to one another and form the polynucleotide. In some embodiments, the methods of the present disclosure also include a step of purifying the formed polynucleotides.

Methods of synthesizing oligonucleotides via new intermediates on a cleavable support having an azidomethyl moiety are disclosed. The method comprises multiple reaction cycles, each of which comprises sequential coupling a nucleoside or oligonucleotide subunit on a cleavable support having an azidomethyl moiety and a nucleoside phosphoramidite or an oligonucleotide phosphoramidite, capping, oxidation / thiolation and deblocking; followed by orthogonal cleavage of the azidomethyl support while keeping all other protecting groups intact. The method can be used in combination with a support moiety for either solid phase or liquid phase oligo synthesis. The soluble support facilitates homogeneous reactions and efficient separations by simple precipitation. The methods also provide novel intermediates useful in the synthesis of oligonucleotide conjugates.

The present application relates to the technical field of nucleotide synthesis, and particularly relates to a 3'-ONH2-deoxyguanosine acid analogue, a preparation method and application thereof, a 7-position nitrogen atom on a base purine ring of the 3'-ONH2-deoxyguanosine acid analogue is substituted by a carbon atom, the change in the structure makes hydrogen bond interaction force between the 3'-ONH2-deoxyguanosine acid analogue and dCTP weaker, the guanosine acid analogue provided by the present application is used to completely replace or partially replace dGTP to reduce the interaction force between GC, effectively reduce the PCR reaction error rate, improve the yield of DNA sequence, and is expected to be applied in gene synthesis, drug preparation and nucleic acid sequence analysis; the preparation method of the 3'-ONH2-deoxyguanosine acid analogue provided by the present application is used for synthesizing 7-deazone modified nucleosides by applying glycosylation reaction, introducing 3-ONH2 modification through twice configuration inversion of 3-position hydroxyl groups on a sugar ring, protecting 3-ONH2 by using acetone, then performing phosphorylation, and finally deprotecting to synthesize the 3'-ONH2-deoxyguanosine acid analogue, the preparation method has mild reaction conditions, is easy to operate, and is beneficial to large-scale production.