An application of a Cas protein, and a method and kit for detecting a target nucleic acid molecule

A detection method and target nucleic acid technology, applied in biochemical equipment and methods, microbial measurement/inspection, genetic engineering, etc., can solve problems such as increasing operational difficulty and achieve high sensitivity

Active Publication Date: 2017-12-19
SHANGHAI TOLO BIOTECH CO LTD
View PDF2 Cites 81 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above method involves the detection of RNA. If only the detection of DNA is r

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • An application of a Cas protein, and a method and kit for detecting a target nucleic acid molecule
  • An application of a Cas protein, and a method and kit for detecting a target nucleic acid molecule
  • An application of a Cas protein, and a method and kit for detecting a target nucleic acid molecule

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0076] Example 1 Cas12a protein detects single-stranded DNA (ssDNA) target (probe FAM label)

[0077] Single-stranded DNA (target-T1-R) is selected as the target sequence to test the response value of different Cas12a proteins to its detection.

[0078] 1. Preparation of crRNA: First, T7-crRNA-F and synthetic oligonucleotide T7-T1-24-R are used to anneal as shown in Table 5 to prepare transcription templates. Specifically, the paired oligonucleotides (4μM) were annealed in 1×PCR buffer (Transgen Biotech) in a total volume of 50μL, and then an annealing procedure was performed: initial denaturation at 95°C for 5 minutes, and then cooling from 95°C to At 20°C, use a thermal cycler to reduce 1°C per minute. CrRNA was synthesized using T7 High Yield Transcription Kit, and the reaction was carried out at 37°C overnight (about 16 hours). Then the RNA was purified using the RNA purification and concentration kit, and quantified with NanoDrop 2000C (Thermo Fisher Scientific), diluted to ...

Example Embodiment

[0081] Example 2 Cas12a protein detects the target of single-stranded DNA (ssDNA) (the probe is double-labeled with HEX and BHQ1)

[0082] Single-stranded DNA (target-T1-R) is selected as the target sequence to test the response value of different Cas12a proteins to its detection.

[0083] 1. Preparation of crRNA: First, T7-crRNA-F is annealed with synthetic oligonucleotide T7-T1-24-R (Table 5) to prepare transcription template. Specifically, the paired oligonucleotides (4μM) were annealed in 1×PCR buffer (TransgenBiotech) with a total volume of 50μL, and then an annealing procedure was performed: initial denaturation at 95°C for 5 minutes, and then cooling from 95°C to 20 ℃, use a thermal cycler to reduce 1 ℃ per minute. CrRNA was synthesized using T7 High Yield Transcription Kit, and the reaction was carried out at 37°C overnight (about 16h). Then use the RNA purification and concentration kit to purify the RNA, and quantify it with NanoDrop2000C, dilute it to a concentration o...

Example Embodiment

[0086] Example 3 Cas12a protein detects double-stranded DNA (dsDNA) target

[0087] Select double-stranded DNA (target-T1) as the target sequence to test the response value of different Cas12a proteins to its detection.

[0088] 1. Preparation of crRNA: First, T7-crRNA-F is annealed with synthetic oligonucleotide T7-T1-24-R (Table 5) to prepare transcription template. Specifically, the paired oligonucleotides (4μM) were annealed in 1×PCR buffer (TransgenBiotech) with a total volume of 50μL, and then an annealing procedure was performed: initial denaturation at 95°C for 5 minutes, and then cooling from 95°C to 20 ℃, use a thermal cycler to reduce 1 ℃ per minute. CrRNA was synthesized using T7 High Yield Transcription Kit, and the reaction was carried out at 37°C overnight (about 16h). Then use the RNA purification and concentration kit to purify the RNA, and quantify it with NanoDrop2000C, dilute it to a concentration of 10μM and store it in the refrigerator at -80°C.

[0089] 2. C...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

An application of a Cas protein, and a method and kit for detecting a target nucleic acid molecule are provided. The method includes adding a guide RNA, Cas12a and a nucleic acid probe into a reaction system containing a target nucleic acid molecule to be detected, and performing detection after a reaction is finished. Through the method, whether a sample contains a target nucleic acid sequence or not can be detected rapidly. Through combination with a nucleic acid amplification technique, the sensitivity of the method is greatly increased. According to characteristics of the method, the method is named as HOLMES (one HOur Low-cost Multiplex Efficient Simple assay), namely an assay method that is low-cost, multiplex, efficient and simple and can be achieved in about one hour. The method can be used for rapidly detecting pathogenic microorganisms, gene mutations, specific target DNAs, and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to a method for detecting target nucleic acid molecules. Background technique [0002] Specific detection of nucleic acid molecules (Nucleic acid detection) method has important application value, such as detection of pathogens, genetic disease detection and so on. In terms of pathogen detection, since each pathogenic microorganism has its unique characteristic nucleic acid molecular sequence, nucleic acid molecular detection for specific species can be developed, also known as nucleic acid diagnostics (NADs, nucleic acid diagnostics), in food safety, environment Microbial contamination detection, human pathogen infection and other fields are of great significance (O'Connor and Glynn 2010, Scheler, Glynn et al. 2014). Another aspect is the detection of single nucleotide polymorphisms (SNPs, single nucleotide polymorphisms) in humans or other species. Understandi...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12Q1/68
CPCC12Q1/6816C12Q2521/327C12Q2525/161C12Q2563/107C12Q1/6823C12N2310/20C12Q1/683Y02A50/30C12Q2521/301C12Q2522/101C12N9/22C12N15/113C12Q1/6886
Inventor 成秋香
Owner SHANGHAI TOLO BIOTECH CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap