8 results about "Reverse Transcription Process" patented technology

A 2-bromoacrylamide assisted cyclization sequencing (BACS) method allows quantitative profiling of pseudouridine (psi) at single base resolution. Based on bromoacrylamide cyclization chemistry, BACS induces psi-to-C mutations during reverse transcription (RT) instead of truncation or deletion of features, thus providing higher resolution and enabling more accurate quantification of psi stoichiometry compared to CMC and BS based methods. The BACS of the invention allows for precise identification of psi positions, in particular in densely modified psi regions and continuous uridine sequences, compared to known methods. Accordingly, methods, compositions, and kits for detecting psi are possible. In the case of RNA molecules, this allows psi to be detected and sequenced in such sequences.

The use of different primer sets in reverse transcription incorporates tags allowing for the selective amplification of cDNA transcribed using the different primers. Primers targeting non-desired RNA sequences such as ribosomal RNA can be used to prevent subsequent amplification of cDNA transcribed from those non-coding fragments. Accordingly effective depletion of non-desired sequences after cDNA amplification can be achieved. Systems and methods of the invention have applications in whole-transcriptome analysis. Non-coding sequence targeting primers can include nucleotide analogs that, when enzymatically processed, prevent subsequent amplification. Library preparation can include single primer isothermal amplification (SPIA) techniques wherein an RNA sequence required for SPIA is included in random primers but is absent from primers targeting non-coding RNA.

Methods for improving complementary DNA (cDNA) yield from a ribonucleic acid (RNA) template, e.g., present in a mixture, during reverse transcription by introducing an anionic polymer are described, including methods for enhancing the ability of the RT to amplify a target low-abundance RNA in a sample, e.g., a complex sample.

The present application provides a kit and method for detecting RNA at room temperature. The kit for detecting RNA at room temperature comprises: a target RNA recognition and amplification component and a double signal amplification component, and the further amplification and reporting of the signal are completed by coupling the two components, and the high-sensitivity detection of RNA is realized. The method for detecting RNA at room temperature of the present application does not need a complex reverse transcription process, can reduce the complexity of the reaction and the cost of the instrument, and has high detection efficiency.

The invention belongs to the technical field of molecular biology, and particularly relates to template conversion oligonucleotide, a kit and application of the template conversion oligonucleotide. In the first aspect of the invention, a template conversion oligonucleotide is provided, the template conversion oligonucleotide comprises a template conversion region at the 3'end, the template conversion region comprises a plurality of nucleotides, the nucleotides comprise sugars and basic groups, and the basic groups are guanine; wherein in the plurality of nucleotides, the 3 '-hydroxyl site of the glycosyl of the nucleotide at the 3'-terminal is provided with a modification for preventing the 3 '-hydroxyl from forming a phosphodiester bond. TSO of conventional reverse transcription is specifically modified, then conventional reverse transcription processes of single cells, space-time omics and the like are carried out, additional enrichment operation is not needed, and the operation process is simple. Not only is the data utilization rate of single molecule sequencing improved, but also the sensitivity of target gene capture is improved, and background interference is reduced.

Disclosed herein are methods for performing in situ sequencing of RNA transcripts with non-uniform 5′ ends. During reverse transcription (RT) of RNA transcripts, RT enzyme is induced to “template-switch” to a separate oligonucleotide provided as the template for the upstream flanking region. This flanking region is grafted onto the beginning of the cDNA, enabling padlock probe detection, rolling circle amplification, and fluorescent in situ sequencing. Overall, the disclosed method for in situ sequencing can be applicable for analyzing exogenously introduced transcripts (e.g., identifying and determining impact of a perturbation including a CRISPR perturbation or shRNA / siRNA / ASO perturbation), analyzing naturally occurring transcripts (e.g., measuring gene expression, detecting splicing events), and analyzing modified, naturally occurring transcripts (e.g., detecting mutations or gene edits).

Methods are described for enhancing complementary DNA (cDNA) synthesis from a ribonucleic acid (RNA) template during reverse transcription by an enzyme having template switching activity. The methods provide for enhancing the processivity of a reverse transcriptase.

The invention discloses a heat-resistant MLV reverse transcriptase mutant as well as a preparation method and application thereof. The invention discloses an MLV reverse transcriptase mutant with an amino acid sequence as shown in SEQ ID NO.1 at first. The invention further discloses a preparation method and application of the MLV reverse transcriptase mutant. According to the MLV reverse transcriptase mutant, key amino acid sites of wild type MLV reverse transcriptase are modified through site-specific mutagenesis, the thermal stability of the MLV reverse transcriptase in the range of 45-65 DEG C is improved, the optimum temperature in the reverse transcription process is increased to 65 DEG C from 45 DEG C, meanwhile, the low RNase H activity of the MLV reverse transcriptase mutant is kept, degradation of RNA-cDNA hybrid chains is reduced, and the MLV reverse transcriptase mutant has the advantages of being high in yield and high in stability. The technical effects of improving the reverse transcription reaction efficiency, enhancing the reverse transcription capability on a complex secondary structure RNA template and reducing the non-specific reaction are achieved.