39 results about "Oligo dt" patented technology

Disclosed is a method for making full-length CDNA libraries, which is for making libraries of cDNAs having a length corresponding to a full-length of mRNAs and comprises the following steps of; binding a tag molecule to a diol structure present in 5' Cap (7MeGpppN) sites of mRNAs, forming RNA-DNA hybrids by reverse transcription using primers such as oligo dT and the mRNAs connected with the tag molecule as templates, and separating RNA-DNA hybrids carrying a DNA corresponding to a full-length of mRNAs from the RNA-DNA hybrids formed above by using function of the tag molecule. To obtain mRNA connected with a tag molecule, the diol structure present in 5' Cap site of mRNA is subjected to a ring-open reaction by oxidation with sodium periodate to form a dialdehyde and the dialdehyde is reacted with a tag molecule having a hydrazine terminus. According to the present invention, there are provided a novel method capable of efficiently labeling 5' Cap site and a method for making full-length cDNA libraries utilizing the labeling method.

Described are methods for production of RNA transcripts using a non-amplified, linearized DNA template in an in vitro transcription reaction. Enzymatic 5′ capping and oligo dT purification can also be included in the methods.

The invention relates to the field of molecular biology and provides Oligo dT primer, wherein Oligo dT primer is a single-stranded DNA molecule containing continuous dT sequences and recognition sites for cutting. The invention further provides a method for constructing a cDNA library on the basis of the Oligo dT primer. The Oligo dT primer is excellent in applicability, can be applied to various different library schemes, and can further accurately locate the initiation site of mRNA molecule in poly A tail. The method for constructing the cDNA library can ensure that all mRNA molecules in a sample can show specificity in the constructed cDNA library.

The invention discloses a three-generation library constructing and sequencing method for overall length amplification of a BCR immunity group library. The method comprises the steps of (1) designingand synthesizing primers according to the constant region of a BCR consistency sequence; (2) synthesizing a first chain cDNA under the action of an Oligo dT primer and a BCR Template Switching Oligo primer; (3) performing first-round amplification and second-round amplification on the overall length of BCR cDNA; (4) mixing samples of BCR overall length amplicon fragments; (5) performing library construction; and (6) performing three-generation sequencing. According to the method disclosed by the invention, multiple primers are designed in the conservative region of a BCR constant region, so that the overall length sequences of the whole BCR are covered. During BCR sequence amplification, in order to further improve the validity of 3' end amplification, multiple groups of semi-nest primersare designed, and through matching and screening of the semi-nest primers, amplicon fragments covering the whole BCR overall length can be obtained. Through a PacBio library constructing technologicalprocess, sequencing contacts having barcode are added to the two ends of each amplicon, and then PacBio sequencing can be performed; and through splitting and data correction, HiFi Reads of which theaccuracy rate is 99% or above can be obtained.

The invention discloses a method for detecting tumor microenvironment-associated antigen expression. The method comprises the following steps of (1) extracting sample RNAs from solid tumor tissue samples; (2) checking the sample RNAs; (3) taking the extracted and qualified sample RNAs as templates, and carrying out inverse transcription of the RNAs by employing oligo dT as a primer to obtain tissue sample cDNAs; and (4) designing a qPCR detection primer, amplifying the tissue sample cDNAs through qPCR amplification and collecting fluorescence signals. The method has the beneficial effects that an inventor selects multiple tumor microenvironment-associated antigens, and correspondingly designs multiple antigen primers; and the expression conditions of the antigens in the tissue samples are specifically detected through current main gene expression analysis method and a fluorescent quantitative PCR method. Through the method, the microenvironment-associated antigen expression values of tumor patients are obtained and the expression values have high reference when a doctor provides the patients with targeted therapeutic schedules.

The invention relates to a method for detecting a PAS (Poly A Site) of an RNA (Ribose Nucleic Acid), and provides a method for analyzing APA (Alternative Polyadenylation) based on 3T-seq (TT-mRNA Terminal Sequencing) within a whole-genome range. The method for analyzing APA comprises the following steps: preparing magnetic beads containing oligo dT, i.e., mixing an oligo dT primer modified by 5' end biotin and the magnetic beads with streptavidin; mixing the magnetic beads with total RNA and screening RNA containing Poly A; performing reverse transcription, synthesizing the second strand of a double-stranded cDNA and breaking the double-stranded cDNA; removing the Poly A structure; after terminal modification, connecting a sequencing linker; and recovering libraries. According to the method for analyzing APA, RNA horizontal operation is reduced, a Poly A sequence is horizontally removed from the DNA, and the influence of the Poly structure on sequencing is eliminated; double-stranded DNA breaking enzyme is selected for treatment, and on the premise that the quality of the libraries is not affected, the DNA is broken horizontally; the experiment process is simplified, the experiment difficulty and cost are lowered and the method for analyzing APA is wide in application range.

The invention discloses a broccoli Ogu cytoplasm quick identification kit and a detection method thereof. The kit comprises a reagent A and a reagent B, wherein the reagent A contains a PCR (Polymerase Chain Reaction) buffer solution, Taq polymerase, dNTP and an Oligo-dT primer, and the reagent B is standard DL-2000DNA Ladder. The detection method comprises the following steps of: DNA extraction of a sample; (1) genome DNA extraction of the sample; (2) PCR amplification reaction; (3) agarose gel electrophoresis detection by which whether the sample is Ogu cytoplasm or not is determined according to the existence/absence of 392bp specific amplification bands. By using the broccoli Ogu cytoplasm quick identification kit, the steps from DNA extraction to the obtaining of detection results can be finished only in several hours, which is very convenient and quick; and by the broccoli Ogu cytoplasm quick identification kit, screening and identifying can be finished in seedling stage, thereby greatly shortening identification cycle. The broccoli Ogu cytoplasm quick identification kit has very important value in broccoli breeding practices.

The invention discloses a method for constructing a circRNA high-throughput sequencing library of an FFPE (formalin-fixed and paraffin-embedded) sample. The method concretely comprises the following steps: (1) extracting total RNA from paraffin sections; (2) adding a PolyA end to the sample RNA; (3) capturing and separating linear RNA by Oligo dT magnetic beads; (4) further removing residual linear RNA; and (5) constructing the circRNA high-throughput sequencing library. The FFPE sample RNA has the characteristics of damages, low content and poor integrity, and the method for constructing thecircRNA high-throughput sequencing library of the FFPE sample has the advantages of simplicity in operation, short time, low cost, stableness in obtaining the library meeting high-throughput sequencing requirements, and avoiding of the problems of large sample size, high rRNA removal cost and low efficiency in the traditional library constructing methods.

The invention relates to the field of molecular biology and provides an Oligo dT primer. The Oligo dT primer contains single-chain DNA molecules having a continuous dT sequence, wherein the DNA molecules contain recognition sites used for cutting; and the recognition sites used for cutting refer to uridine monophosphate, deoxyinosine or phosphorothioate bond. The invention also provides a method for constructing a cDNA library on the basis of the Oligo dT primer. The Oligo dT primer disclosed by the invention is high in applicability and can be applied to multiple different library construction schemes; the initiation sites at the polyA tails of mRNA molecules can be accurately positioned; and according to the method for constructing the cDNA library disclosed by the invention, all the mRNA molecules in samples can be specifically reflected in the constructed cDNA library.

The present invention includes a method and kit for cDNA synthesis of a 3′UTR/poly(A) tail junction of cellular RNA comprising: obtaining RNA comprising a 3′UTR/poly(A) junction and a poly(a) tail; combining the RNA with three terminating nucleotides of modified-deoxyGTP, modified-deoxyCTP and modified-deoxyATP, dNTPs, and adaptor sequence-oligo-dT; performing reverse transcription of the RNA with a reverse transcriptase primed with the adaptor sequence-oligo-dT to form terminated cDNA fragments that are stochastically terminated upstream of the 3′UTR/poly(A) junction, but not within the poly(A) tail; isolating the terminated cDNA fragments; chemically ligating a functionalized 5′ adaptor to the terminated cDNA; and amplifying the chemically-ligated cDNA into an amplification product, wherein the cDNA is enriched for sequences at the 3′UTR/poly(A) tail junction without fragmentation or enzymatic ligation.

The invention relates to a gene sequence of a sucrose synthetase gene Ac-Sy1 in pineapple fruits and a protein sequence encoded by the same, belonging to the technical of biology. The invention further relates to a cloning method of the gene, which comprises the following steps of: extracting the total RNA in the pineapple fruits by using a modified SDS method; detecting the total RNA in the pineapple fruits by using agarose gel electrophoresis; carrying out reverse transcription to synthesize a first chain cDNA by using the total RNA as a template and using Oligo dT as a primer for; using the cDNA as a template and carrying out PCR amplification by using the Ex-Taq of Takara; connecting a PCR product with a pGEM-T easy Vector, converting, screening the positive for cloning, further validating by PCR and sequencing to obtain the Ac-Sy1 gene. The gene can provide the theoretical evidence for scientifically adjusting and controlling the quality of the pineapple fruits.

The invention provides a direct plant mRNA (messenger ribonucleic acid) extraction method, belongs to the biomolecular level technology field, and particularly relates to a method for extracting plant mRNA directly.The direct plant mRNA extraction method is provided to solve the problems of process complexity and low efficiency of conventional plant mRNA extraction methods.A new technique of directly extracting solid-phase mRNA genes is provided, Oligo dT(15-25) is adsorbed on the head surface of an intradermal needle after a series of treatments including washing, incubating, culturing, embedding and drying of the intradermal needle, the treated intradermal needle is punched at a set position of a plant material to extract mRNA of the plant material.By the direct plant mRNA extraction method, plant mRNA extraction is realized, the mRNA can be extracted from the plant material in 2 minutes, and simplicity, convenience and rapidness of the operation process are achieved.

The invention discloses a preparation method and application of microspheres for purifying mRNA (messenger Ribonucleic Acid), and the preparation method of the microspheres for purifying mRNA comprises the following steps: S1) washing polystyrene microspheres with active groups on the surface by using a coupling buffer solution; s2) Oligo dT is added to react with the polystyrene microspheres, and after the reaction is finished, a coupling buffer solution is used for cleaning; and S3) adding a blocking reagent for reaction, and after the reaction is finished, cleaning with a coupling buffer solution to prepare the microspheres for purifying mRNA. The surface of the rigid polystyrene microsphere is modified with amino, carboxyl, epoxy group, active ester (NHS) or benzenesulfonyl, so that the rigid polystyrene microsphere has reaction activity and can quickly react with Oligo dT, and Oligo dT is fixed on the surface of the microsphere, so that the microsphere has mRNA separation and purification capabilities; and the microspheres have rigid structures, so that the microspheres can bear a certain pressure, and the Oligo dT microspheres are loaded into a chromatographic column, so that mRNA can be rapidly separated and purified on equipment such as liquid chromatography and the like.

The invention discloses a preparation method and application of magnetic microspheres for purifying mRNA (messenger ribonucleic acid). The preparation method of the magnetic microspheres for purifying mRNA comprises the following steps: S1) washing the magnetic microspheres with active groups on the surfaces by using a coupling buffer solution; s2) Oligo dT is added to react with the magnetic microspheres, and then a coupling buffer solution is used for cleaning; and S3) adding a blocking reagent for reaction, and after the reaction is finished, cleaning with a coupling buffer solution to prepare the magnetic microspheres for purifying mRNA. The surface of the magnetic microsphere is modified with amino, carboxyl, epoxy group, active ester (NHS) or benzenesulfonyl, so that the magnetic microsphere has reaction activity and can quickly react with Oligo dT, and Oligo dT is fixed on the surface of the magnetic microsphere, so that the magnetic microsphere has mRNA separation and purification capabilities, and because the magnetic microsphere has magnetism, in the presence of a magnetic field, the mRNA separation and purification capability of the magnetic microsphere is greatly improved. The magnetic microspheres can quickly move towards one side of a magnetic field, so that mRNA can be quickly separated and purified.