321 results about "Stem-loop" patented technology

The present teachings provide methods, compositions, and kits for reverse transcribing and amplifying small nucleic acids such as micro RNAs. High levels of multiplexing are provided by the use of a zip-coded stem-loop reverse transcription primer, along with a PCR-based pre-amplification reaction that comprises a zip-coded forward primer. Detector probes in downstream decoding PCRs can take advantage of the zip-code introduced by the stem-loop reverse transcription primer. In some embodiments, further amplification is achieved by cycling the reverse transcription reaction. The present teachings also provide compositions and kits useful for performing the reverse transcription and amplification reactions described herein.

Provided are tricyclo-DNA (tc-DNA) AON and methods employing tc-DNA AON for modifying splicing events that occur during pre-mRNA processing. Tricyclo-DNA (tc-DNA) AON are described that may be used to facilitate exon skipping or to mask intronic silencer sequences and/or terminal stem-loop sequences during pre-mRNA processing and to target RNase-mediated destruction of processed mRNA. Tc-DNA AON described herein may be used in methods for the treatment of Duchenne Muscular Dystrophy by skipping a mutated exon 23 or exon 51 within a dystrophin gene to restore functionality of a dystrophin protein; in methods for the treatment of Spinal Muscular Atrophy by masking an intronic silencing sequence and/or a terminal stem-loop sequence within an SMN2 gene to yield modified functional SMN2 protein, including an amino acid sequence encoded by exon 7, which is capable of at least partially complementing a non-functional SMN1 protein; and in methods for the treatment of Steinert's Myotonic Dystrophy by targeting the destruction of a mutated DM1 mRNA comprising 3′-terminal CUG repeats.

The invention relates to construction of rice transgenic materials and aims to provide a gene editing method for knocking out rice MIRNA393b stem-loop sequences with application of a CRISPR(clustered regulatory interspersed short palindromic repeat)-Cas9 system. The gene editing method comprises steps as follows: gRNA target sites are selected for cloning and GG linking, enzyme digestion is performed after amplification, and a product is linked with a pGREB 32 vector; escherichia coli competent cells are transformed; plasmids with a correct sequencing result are used for transforming agrobacteria, transgenic plants are obtained through mediated transformation of rice calli, and transgenic positive lines are obtained; the T0-generation mutant plant seeds are collected for seeding, and the T1-generation plants are subjected to homozygote screening; homozygous lines which are discovered to be negative through MIRNA393b expression are rice mutants completely losing the MIRNA393b stem-loop sequences and MIRNA393b stem-loop sequence expression. According to the gene editing method, MIRNA stem-loop sequences can be effectively knocked out, and loss-of-function mutants of different members in the same MIRNA family can be prepared; the mutant plant propagates to obtain a large number of seeds and is an ideal material for acquiring rice MIRNA393b gene functions successfully.

The present teachings provide methods, compositions, and kits for quantifying target polynucleotides. In some embodiments, a reverse stem-loop ligation probe is ligated to the 3′ end of a target polynucleotide, using a ligase that can ligate the 3′ end of RNA to the 5′ end of DNA using a DNA template, such as T4 DNA ligase. Following digestion to form an elongated target polynucleotide with a liberated end, a reverse transcription reaction can be performed, followed by a PCR. In some embodiments, the methods of the present teachings can discriminate between polymorphic polynucleoitides that vary by as little as one nucleotide.

Disclosed herein include systems, methods, compositions, and kits for molecular barcoding on the 5′-end of a nucleic acid target. After barcoding a nucleic acid target using an oligonucleotide barcode comprising a target binding region and a molecular label to generate a barcoded nucleic acid molecule, an oligonucleotide comprising a complement of the target binding region can be added to generate a barcoded nucleic acid molecule comprising the target-binding region and the complement of the target-binding region. A stem loop is formed with intra-molecular hybridization of the barcoded nucleic acid molecule, which can be extended to generate an extended barcoded nucleic acid molecule comprising the molecular label and a complement of the molecular label.

The application relates generally to methods useful for the selective amplification of one or more target nucleic acid or fragments thereof, as well as compositions and kits comprising said amplification reaction mixtures. More specifically, the application relates to a composite primer that comprises a 5′ promoter portion and a 3′ target-recognition portion which is complementary to the 3′ end portion of a target polynucleotide sequence; and optionally, a means for identifying the 5′ end portion of the target polynucleotide sequence. The amplification reaction mixture comprises at least one handle-stem-loop structure which comprises a 5′ single-stranded handle comprising the promoter portion and a double-stranded stem comprising at least one pair of self-folding segments hybridized to each other, and optionally, a single-stranded loop comprising the sequence between the pair of self-folding segments.

Recombinant nucleic acid molecules are provided that form hair pin structures and can be used to down-regulate gene expression. For example, a nucleic acid molecule can comprise a flanking and lower stem loop sequence from a mir-16 gene; an antisense target sequence; a mir-30 loop sequence; a complement of the anti-sense target sequence; and a lower stem loop complementary to the mir-16 sequence. Methods for down regulating gene expression in a cell using such recombinant nucleic acid molecules are also provided.

Dual nucleic acid probes with resonance energy transfer moieties are provided. In particular, fluorescent or luminescent resonance energy transfer moieties are provided on hairpin stem-loop molecular beacon probes that hybridize sufficiently near each other on a subject nucleic acid, e.g. mRNA, to generate an observable interaction. The invention also provides lanthanide chelate luminescent resonance energy transfer moieties on linear and stem-loop probes that hybridize sufficiently near each other on a subject nucleic acid to generate an observable interaction. The invention thereby provides detectable signals for rapid, specific and sensitive hybridization determination in vivo. The probes are used in methods of detection of nucleic acid target hybridization for the identification and quantification of tissue and cell-specific gene expression levels, including response to external stimuli, such as drug candidates, and genetic variations associated with disease, such as cancer.

There is provided a method for detecting a target RNA molecule in a sample. The method comprises reverse transcribing the target RNA contained in the sample using an RT oligonucleotide, the RT oligonucleotide comprising a stem-loop portion containing one or more nucleotides modified or modifiable to block DNA polymerase extension and a target annealing portion that is complementary to a downstream portion of the target RNA, the target annealing portion located 3′ to the stem-loop portion, to produce a reverse transcription product that comprises the RT oligonucleotide and a 3′ extended region; amplifying the reverse transcription product using (i) a first amplification primer that anneals to a downstream portion of the 3′ extended region of the reverse transcription product and (ii) a second amplification primer that anneals to an interface portion of a DNA strand complementary to the reverse transcription product, the interface portion comprising a region that is complementary to a 3′ portion of the RT oligonucleotide and a 5′ portion of the 3′ extended region in the reverse transcription product, to produce an amplification product; and detecting the amplification product; wherein the stem-loop portion adopts a stem-loop structure under conditions used for said reverse transcribing but does not adopt the stem-loop structure under conditions used for said amplifying and wherein when the stem-loop portion contains one or more nucleotides that are modifiable to block DNA polymerase extension, the method further comprises modifying the modifiable nucleotide prior to said amplifying.

The present invention discloses a 17beta-estradiol visualization detection method based on DNA nano-structure, and a 17beta-estradiol visualization detection kit based on DNA nano-structure. The working principle is that 17b-estradiol interacts with aptamer, a strand displacement reaction is started, three groups of stem-loop DNA probes are constantly opened to form DNA nano-structures, a G tetramer having catalysis activity is formed on the terminals of the three DNA arms in the DNA nano-structures, the G tetramer is bound with hemin so as to form a compound having catalysis activity and similar to HRP, TMB is subjected to catalytic oxidation, and a blue substrate is produced, wherein the result is visible, and the concentration of 17b-estradiol is directly related to the blue shade. According to the present invention, the operation is simple, the whole reaction can be completed at the room temperature, the high sensitivity is provided, the detection limit on 17b-estradiol is 100 fM, the good specificity is provided, the detection result is directly visible without any detection equipment, and the method and the kit can be used for the on-site rapid detection of 17b-estradiol.

The invention provides sense antiviral compounds and methods of their use in inhibition of growth of viruses of the Flaviviridae, Picornoviridae, Caliciviridae, Togaviridae, Coronaviridae families and hepatitis E virus in the treatment of a viral infection. The sense antiviral compounds are substantially uncharged morpholino oligonucleotides having a sequence of (12-40) subunits, including at least (12) subunits having a targeting sequence that is complementary to a region associated with stem-loop secondary structure within the 3′-terminal end (40) bases of the negative-sense RNA strand of the virus.

The present invention discloses a new precursor miRNA and applications of the new precursor miRNA in tumor treatment. Specifically the present invention provides a precursor miRNA, wherein the structure represented by a formula I exists at the 5' to 3' terminal, B1 is anti-miRNA-214-5p, B2 is a sequence substantially complementary or completely complementary to B1, B2 and C are not complementary, C is a stem-loop structural sequence, A1 and A2 respectively are none, optionally RNA sequences comprising 4-5 bases, the precursor miRNA can be processed in a host to form anti-miRNA-214, and in the anti-miRNA-214, only the anti-miRNA-214-5p is expressed while the anti-miRNA-214-3p is not expressed. The formula I is defined in the specification.

A method for detection of nucleic acid targets using as reagents: (1) a stem loop probe having a long strand for hybridizing to a target, a short strand, usually with a free 3'-end for extension, hybridized to a portion of the long strand and a linker forming a loop and joining the short and long strands; and (2) a hybridizing reagent that hybridizes to the short strand when released by the target. The target is detected by extension of one or both the probe or hybridizing reagent along each other. A circular hybridizing reagent can be employed with DNA polymerase for concatenated extension of the probe 3'-end or extension of the 3'-end of the probe along a hybridizing reagent having a promoter sequence for forming transcripts of at least a portion of the long strand or the hybridizing reagent. A non-cleavable restriction site consensus sequence in the linker, where the hybridizing reagent is extended with dNTPs and DNA polymerase and the extended sequence is cleaved with a restriction enzyme, so that chains may be repetitively produced and cleaved. The presence of the target nucleic acid is established by detecting the concatenated chain, the RNA transcripts or the repetitively produced chains.

The invention relates to a method for detecting miRNA (micro Ribose Nucleic Acid) by an improved stem-lop primer qRT-PCR (Quantitative Reverse Transcription Polymerase Chain Reaction). The method comprises the steps: (1), designing a miRNA detection primer, wherein a stem-loop primer comprises two parts of a general stem loop sequence and a specific primer sequence which is in complementary pairing with a 3' end of a target miRNA, a base number which is in complementary pairing with the target miRNA is 11bp, a downstream primer needle of qPCR is specific to a general stem loop sequence, an upstream primer needle is specific to a 5' end of the target miRNA, a GC tail is added in the 5' end of the upstream primer so that the annealing temperatures of the upstream primer and the downstream primer are close; (2), performing inverse transcription on the target miRNA into cDNA (complementary Desoxvribose Nucleic Acid); and (3), performing a quantitative PCR amplification and detecting by adopting an SYBR Green qPCR detection method. The method disclosed by the invention is good in specificity, and high in sensitivity; and one downstream primer is only synthesized, and an SYBR Green I fluorochrome method is adopted, and thus the cost is greatly saved.

The invention belongs to the field of biology, and relates to an application of serum ssc-miR-215 as a molecular marker on detecting stress injury of the intestinal tract of a pigling. A nucleotide sequence of serum ssc-miR-215 is represented as SEQ ID NO.1. A nucleotide sequence of a stem-loop primer required by reverse transcription of ssc-miR-215 is represented as SEQ ID NO.2. A nucleotide sequence of a forward primer required by augmentation of ssc-miR-215 is represented as SEQ ID NO.3. Diagnosis of stress injury of the intestinal tract of a weaned pigling is realized by adopting the above primers. A diagnostic method comprises extraction of serum total RNA, separation of small molecular RNA, synthesis of cDNA, and augmentation and authentication of PCR. The invention provides guidance for prevention, diagnosis and treatment of stress injury of the intestinal tract of a pigling, and provides a novel molecular marker for marker assisted selection of an anti-stress pig line.

The invention discloses a quantitative biomolecule detection method comprising the following steps of: firstly, designing fluorescently-labeled H1* and H2* probes according to a target biomolecule sequence; secondly, heating solution with set concentration of fluorophore-labeled stem-loop-structure nucleic acid probes H1* and H2* on PCR (Polymerase Chain Reaction) instrument for 2 minutes at 95 DEG C, and then placing the solution away from light for 1 hour to 2 hours at a room temperature so as to allow a stem-loop structure to close fully; thirdly, preparing solution with set specification concentration from target biomolecules, respectively adding the biomolecule solution with different specification concentration and the H1* and H2* solution in a plurality of centrifuge tubes, and performing HCR (Hybridization Chain Reaction) on the biomolecule solution and the H1* and H2* solution which are mixed uniformly at the room temperature; after the HCR is finished, adding a proper amount of carbon materials in the mixed solution according to the concentration of the H1* and H2* in the second step, reacting the solution for 30 minutes at a room temperature, and detecting and recording fluorescent signals of the system; and finally, performing quantitative analysis on the target biomolecules by virtue of the fluorescent signals. According to the quantitative biomolecule detection method, the cost is low, the sensitivity is high, and the operation is simple.

The new quantitative PCR detection process of micro RNAs (microRNAs) and small interference RNAs (siRNAs) includes: complementing the bases near the 5' end and the 3' end of retroprimer to form a stem-loop structure, combining the 3' end of retroprimer and target nucleic acid complementarily, and performing reverse transcription reaction with reverse transcriptase; amplifying cDNA obtained through the reverse transcription reaction with partial sequence of the retroprimer as one of the PCR primers; and real-time monitoring quantitative PCR with fluorescence labeled molecular beacon probe.

The invention discloses a microRNA biomarker. The microRNA biomarker is one of miR-21, miR-25, miR-29c, miR-93, miR-198, miR-221 and miR-222 the sequences of which are correspondingly shown in SEQ ID NO:1-SEQ ID NO:7. The invention also discloses an application of the microRNA biomarker in preparing a kit capable of detecting liver cancer. The invention simultaneously discloses a kit capable of detecting liver cancer. The kit consists of a reverse transcription system, an amplification system and a primer system, wherein the primer system comprises the stem-loop primer and amplification primer of one of the miR-21, the miR-25, the miR-29c, the miR-93, the miR-198, the miR-221 and the miR-222. The invention can be used for screening and diagnosing the liver cancer.

The invention discloses a bisphenol A detecting method and detecting kit based on the dual-quenching-group nucleic acid self-assembling technology. Two DNAs of stem loop structures are designed, wherein one DNA of the stem loop structure is used for modifying a fluorophore, and the other DNA of the stem loop structure is used for modifying two quenching groups; and under the condition that bisphenol A exists, loop opening hybridization can be continuously carried out on the two DNAs of the stem loop structures, so that the fluorophore and the quenching groups get close to each other, fluorescence energy transferring happens, and fluorescence is accordingly quenched. According to the detecting method, signal amplification is sourced from continuous loop opening complementation of the two DNAs of the stem loop structures, and no protease is needed to be used, so that operation is easy, a reaction can be completed at the room temperature, the sensitivity is high, and the selectively is good. The two quenching groups exist on one DNA of the stem loop structure at the same time, and the fluorophore on the other DNA of the stem loop structure can be quenched more effectively, so that the fluorescence background is effectively reduced, the detection sensitivity is greatly improved, and the detection limit is 0.4 pM.

The invention provides a mercuric ion detection kit based on constant-temperature cascading nucleic acid amplification and a detection method thereof. Thymine-containing DNA identification elements are specifically bound with mercuric ions and then are folded to form an intra-molecular stem-loop structure; The 3' terminal of the DNA can be identified by polymerase, the DNA itself serves as a template to start strand displacement amplification reaction so as to produce a large amount of single-stranded DNA products; the single-stranded DNA products can open a restriction enzyme cutting site containing molecular beacon stem-loop structure to produce fluorescence signals; at the same time, the single-stranded DNA products can be also used as primers, and hybridization combined molecular beacons are used as templates to trigger secondary strand displacement amplification reaction, and released SDA products can form heteroduplexes with new molecular beacons so as to produce cascaded-amplified fluorescent signals. The detection method is high in sensitivity, ingeniously achieves cascading amplification of strand displacement amplification reaction without system complexity increase, is high in amplification efficiency and response speed and can achieve mercuric ion quantification within 30 minutes, and the detection limit is as low as 2 nM.