1254 results about "Base J" patented technology

Based on the above and on the remarkable properties of the 2′-O,4′-C-methylene bridged LNA monomers it was decided to synthesise oligonucleotides comprising one or more 2′-O,4′-C-methylene-β-D-xylofuranosyl nucleotide monomer(s) as the first stereoisomer of LNA modified oligonucleotides. Modelling clearly indicated the xylo-LNA monomers to be locked in an N-type furanose conformation. Whereas the parent 2′-deoxy-β-D-xylofuranosyl nucleosides were shown to adopt mainly an N-type furanose conformation, the furanose ring of the 2′-deoxy-β-D-xylofuranosyl monomers present in xylo-DNA were shown by conformational analysis and computer modelling to prefer an S-type conformation thereby minimising steric repulsion between the nucleobase and the 3′-O-phopshate group (Seela, F.; Wömer, Rosemeyer, H. Helv. Chem. Acta 1994, 77, 883). As no report on the hybridisation properties and binding mode of xylo-configurated oligonucleotides in an RNA context was believed to exist, it was the aim to synthesise 2′-O,4′-C-methylene-β-D-xylofuranosyl nucleotide monomer and to study the thermal stability of oligonucleotides comprising this monomer. The results showed that fully modified or almost fully modified Xylo-LNA is useful for high-affinity targeting of complementary nucleic acids. When taking into consideration the inverted stereochemistry at C-3′ this is a surprising fact. It is likely that Xylo-LNA monomers, in a sequence context of Xylo-DNA monomers, should have an affinity-increasing effect.

The present invention provides a polynucleotide that not only has a high RNA interference effect on its target gene, but also has a very small risk of causing RNA interference against a gene unrelated to the target gene. A sequence segment conforming to the following rules (a) to (d) is searched from the base sequences of a target gene for RNA interference and, based on the search results, a polynucleotide capable of causing RNAi is designed, synthesized, etc.:

• (a) The 3′ end base is adenine, thymine, or uracil,
• (b) The 5′ end base is guanine or cytosine,
• (c) A 7-base sequence from the 3′ end is rich in one or more types of bases selected from the group consisting of adenine, thymine, and uracil, and
• (d) The number of bases is within a range that allows RNA interference to occur without causing cytotoxicity.

This application relates to monomers of the general formula (I) for the preparation of PNA (peptide nucleic acid) oligomers and provides method for the synthesis of both predefined sequence PNA oligomers and random sequence PNA oligomers:

wherein

• R1, R2, R3, R4, R5 is independently H, halogen, C₁–C₄ alkyl, nitro, nitrile, C₁–C₄ alkoxy, halogenated C₁–C₄ alkyl, or halogenated C₁–C₄ alkoxy, wherein at least one of R1, R3, and R5 is nitro;
• R6 is H or protected or unprotected side chain of natural or unnatural α-amino acid; and
• B is a natural or unnatural nucleobase, wherein when said nucleobase has an exocyclic amino function, said function is protected by protecting group which is labile to acids but stable to weak to medium bases.

Convenient techniques for discriminating the base type in a base sequence of a nucleic acid are provided. The technique includes the step (a) of preparing a sample solution containing a nucleic acid, a primer having a base sequence that includes a complementary binding region which complementarily binds to the nucleic acid, and a nucleotide; the step (b) of allowing the sample solution to stand under a condition to cause an extension reaction of the primer, and producing pyrophosphate when the extension reaction is caused; the step (c) of bringing the sample solution into contact with the front face of a H⁺ hardly permeable membrane having H⁺-pyrophosphatase, which penetrates from front to back of the membrane, of which active site that hydrolyzes pyrophosphate being exposed to the front face; the step (d) of measuring the H⁺ concentration of at least either one of the solution at the front face side of the H⁺ hardly permeable membrane or the solution at the back face side of the H⁺ hardly permeable membrane, in a state where the H⁺-pyrophosphatase is immersed in the solution; the step (e) of detecting the extension reaction on the basis of the result of measurement in the step (d) ; and the step (f) of discriminating the base type in the base sequence of the nucleic acid on the basis of the result of detection in the step (e).

The present invention provides a method for identifying one or more low abundance sequences differing by one or more single-base changes, insertions, or deletions, from a high abundance sequence in a plurality of target nucleotide sequences. The high abundance wild-type sequence is selectively removed using high fidelity polymerase chain reaction analog conversion, facilitated by optimal buffer conditions, to create a restriction endonuclease site in the high abundance wild-type gene, but not in the low abundance mutant gene. This allows for digestion of the high abundance DNA. Subsequently the low abundant mutant DNA is amplified and detected by the ligase detection reaction assay. The present invention also relates to a kit for carrying out this procedure.

The invention provides a gene base editor, in particular relates to fusion protein. The fusion protein is characterized by comprising two fragments. The first fragment comprises APOBEC3A and the second fragment comprises CRISPR-related Cas protein. The base editor is capable of performing base edition in DNA and performing deamination on cytosine to be uracil, and the base editor still has higherediting efficiency even if cytosine is located at a GpC site or in a hypermethylated state.

A method of nucleic acid detection including (a) contacting a primed template with a polymerase, nucleotide cognate of a first base type and nucleotide cognate of a second base type under ternary complex stabilizing conditions; (b) contacting the primed template nucleic acid with a polymerase, nucleotide cognate of the first base type and nucleotide cognate of a third base type under ternary complex stabilizing conditions; (c) examining products of (a) and (b) for signals produced by a ternary complex that includes the primed template nucleic acid, polymerase and next correct nucleotide, wherein signals for the product of (a) are ambiguous for the first and second base type, and signals for the product of (b) are ambiguous for the first and third base type; (d) disambiguating signals acquired in (c) to identify the next correct nucleotide.

The invention provides a method for precisely editing a genome DNA sequence. A TALEN, CRISPR/Cas9 and other gene targeting technologies and homologous recombination technologies are utilized in a combined mode for precisely editing the genome DNA sequence. According to the method, the technical problem that in the prior art, a specific sequence cannot be deleted is solved, the specific base sequence can be seamlessly inserted or deleted in a fixed point mode, and seamless free modification of the genome DNA sequence of eukaryocyte is achieved for the first time.

The invention discloses a method knocking out pig FGL2 genes with CRISPR-Cas9 specificity and sgRNA for specificity targeting FGL2 genes. The target sequence of the sgRNA of the specificity targeting FGL2 genes on the FGL2 genes meets a sequence arrangement rule of 5'-N(20) NGG- 3'; N(20)represents 20 continous basic groups; each N indicates A, T, C or G; the target sequence on the FGL2 is placed in exon encoding areas of the FGL2; and the target sequence on the FGL2 genes are unique. In the method knocking out pig FGL2genes with CRISPR-Cas9 specificity, FGL2genes of the pigs can be quickly, accurately and high-efficiently knocked out in a specific way; and problems of long term and high cost during gene knockout of the FGL2genes can be solved.

The invention provides a new method for DNA sequencing called “natural sequencing by synthesis” (nSBS). According to the method, DNA that includes a desired sequence is synthesized using a dNTP mix with a small percentage of fluorescently-labeled nucleotides. The fluorescent label is cleavable. In contrast to previous methods that utilize 100% labeled nucleic acids, use of a small percentage of labeled nucleic acids minimizes the distortion of the natural structure of the extending DNA strand and the DNA polymerase. Using the disclosed methods with less than 10,000 copies of template DNA and 10% of the nucleotides labeled, long homopolymer stretches up to 20 bases can be sequenced with high accuracy and Q20 (with 99% accuracy) read lengths of up to 1,000 bases can be achieved. A Q20 read length of greater than 100 bases can potentially be achieved, even if the sequencing is performed with 1,000 copies of a template and 10% of the nucleotides labeled.

The invention relates a technique for fast detecting food-borne pathogens based on a loop-mediated isothermal amplification, LAMP technique. A primer for diction of vibrio parahemolyticus can augment the specific base sequence of a target gene which is the tih-GenBank (accession no. AY578148) of the vibrio parahemolyticus, and the primer is complementary to a part of or a complementary chain fragment of the nucleic acid sequence on the 946-1140 loci on the target gene. The invention provides a primer having specificity to a specific gene fragment of the vibrio parahemolyticus, and through detecting whether or not the detecting specimen in a reagent box of the primer unit contains the specific gene fragment of the vibrio parahemolyticus, determines whether the vibrio parahemolyticus exists in the specimen or not.

The invention belongs to the technical field of biology, and specifically relates to Cas9 nuclease and an application. The Cas9 nuclease (Cas9-R919P) has the activity as Cas9 nuclease, fits to a CRISPR/Cas9 system, and is made by mutating the 919 location arginine of wild Cas9 nuclease to proline. A protruding fracture terminal is generated by cutting a DNA double chain with the Cas9 nuclease (Cas9-R919P), and a basic group complementary to the protruding fracture terminal is added in a filling-in manner, so that accurate addition at a specific position of the genome DNA segment can be carried out.

Some aspects of this disclosure provide strategies, systems, reagents, methods, and kits that are useful for the targeted editing of nucleic acids, including editing a single site within the genome ofa cell or subject, e.g., within the human genome. In some embodiments, fusion proteins of Cas9 and nucleic acid editing proteins or protein domains, e.g., deaminase domains, are provided. In some embodiments, methods for targeted nucleic acid editing are provided. In some embodiments, reagents and kits for the generation of targeted nucleic acid editing proteins, e.g., fusion proteins of Cas9 andnucleic acid editing proteins or domains, are provided.

Methods are provided for reducing the complexity of a population of nucleic acids prior to performing an analysis of the nucleic acids, e.g., sequence analysis. The methods result in a subset of the initial population enriched for a desired property, or lacking nucleic acids having an undesired property. The methods are particularly useful for analyzing populations having a high degree of complexity, e.g., chromosomal-derived DNA, whole genomic DNA, or mRNA populations. In addition, such methods allow for analysis of pooled samples.

Provided are methods and systems for detecting formation of nucleotide-specific ternary complexes comprising a DNA polymerase, a nucleic acid, and a nucleotide complementary to the templated base of the primed template nucleic acid. The methods and systems facilitate determination of the next correct nucleotide without requiring chemical incorporation of the nucleotide into the primer. These results can even be achieved in procedures employing unlabeled, native nucleotides.

The invention discloses a CRISPR-Cas9 targeted-knockout human colorectal cancer cell PPP1R1C gene and a specific sgRNA sequence. The invention comprises the following steps: firstly, acquiring sgRNA of a specific targeting PPP1R1C gene functional area, wherein a base sequence of the sgRNA is shown as SEQ ID NO.1; then, constructing an sgRNA-lentiviral vector system of the PPP1RQC gene, wherein thesystem contains Cas9 protein; and finally, infecting human colorectal cancer HT-29 cells with CRISPR/Cas9 lentivirus containing the sgRNA, so that a cell line that a PPP1R1C protein expression levelis obviously reduced is obtained. The invention is simple in operating steps, good in sgRNA targeting performance and high in cutting efficiency on the PPP1R1C gene; the constructed CRISPR/Cas9 lentiviral system is high in knockout efficiency and is capable of achieving specific knockout of the PPP1R1C gene, so that the human colorectal cancer cells that the PPP1R1C gene is knocked out is obtained; therefore, a powerful tool is provided for further researching an action mechanism of the PPP1R1C in the colorectal cancer cells.