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Application of high-temperature resistance Cas protein and detection system and kit for target nucleic acid molecule

A target nucleic acid, kit technology, applied in biochemical equipment and methods, microbial determination/inspection, hydrolase, etc., can solve problems affecting Toehold detection and other issues

Active Publication Date: 2019-12-10
SHANGHAI TOLO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method involves RNA extraction, amplification, and Toehold reaction detection process. By design, if one virus strain happens to be different from another strain at the site sequence of PAM (that is, one is NGG, the other is different) , the PAM-containing strain can be targeted to be cleaved by Cas9, thereby affecting the amplification process and the detection of Toehold

Method used

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  • Application of high-temperature resistance Cas protein and detection system and kit for target nucleic acid molecule
  • Application of high-temperature resistance Cas protein and detection system and kit for target nucleic acid molecule
  • Application of high-temperature resistance Cas protein and detection system and kit for target nucleic acid molecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0294] Embodiment 1Cas12b protein detects the target of double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA)

[0295] Select single-stranded DNA or double-stranded DNA (target T1) as the target sequence, and test the response value of different concentrations of Cas12b protein and sgRNA to its detection.

[0296] 1. Preparation of guide RNA (sgRNA)

[0297] First construct the plasmid pUC18-sgRNA-T1 with pUC18 as the plasmid backbone, which inserts the T7 promoter and the template DNA sequence for transcription of sgRNA on pUC18 (Note: the sgRNA transcribed from this template targets a sequence called T1). The method is to use the pUC18 plasmid as a template, pUC18-1-F and pUC18-1-R as primers, first perform a round of PCR, T4DNA Ligase ligates the PCR product, transforms it into DH10b, and obtains a correct clone by sequencing, which is called pUC18- sgRNA-T1-pre. Then use pUC18-sgRNA-T1-pre as template, pUC18-2-F and pUC18-2-R as primers, carry out the second round ...

Embodiment 2

[0310] Example 2 HOLMES v2.0 (LAMP combined with Cas12b) test response sensitivity

[0311] By detecting the excited fluorescence intensity of the fluorescent probe (HEX-N12-BHQ1), determine the target DNA concentration required for Cas12b to exercise bypass single-stranded DNA cleavage activity, that is, the sensitivity of Cas12b bypass single-stranded DNA cleavage reaction.

[0312] 1. Preparation of sgRNA

[0313] First, using the previously constructed plasmid pUC18-sgRNA-T1 as a template, primers named sgRNA-DNMT1-3-F and sgRNA-DNMT1-3-R were designed, and 20% of the target DNA in the sgRNA targeting T1 was PCR-generated. 1 base was replaced by sgRNA targeting DNMT1-3, and another plasmid pUC18-sgRNA-DNMT1-3 was obtained.

[0314] Then, using the plasmid pUC18-sgRNA-DNMT1-3 as a template, and T7-crRNA-F and ZLsgRNA-DNMT1-3-R as primers, the DNA template required for in vitro transcription was amplified by PCR. Then add DpnI to the PCR product (1 μl DpnI (10U / μl) per 50 ...

Embodiment 3

[0332] Embodiment 3Cas12b test single base mutation target

[0333] Select single-stranded DNA or double-stranded DNA as the target sequence, and test the change of Cas12btrans cutting fluorescence signal when there is a single-base mutation in the target DNA, so as to detect the single-base mutation.

[0334] 1. Preparation of guide RNA (sgRNA)

[0335]Using the plasmid pUC18-sgRNA-DNMT1-3 as a template, T7-crRNA-F as an upstream primer, and oligonucleotides containing guide sequences complementary to different targets as a downstream primer, the DNA required for in vitro transcription was amplified by PCR template. Then add DpnI to the PCR product (1 μl DpnI (10U / μl) per 50 μl PCR system), bathe in water at 37°C for 30 min, digest the plasmid DNA template, and recover the PCR product using the Gel and PCR Product Column Recovery Kit (Promega). product. Using this PCR recovery product as a template, use T7 High Yield Transcription Kit (Thermo) to synthesize full-length sgR...

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Abstract

The invention provides application of high-temperature resistance Cas protein and a detection system and a kit for a target nucleic acid molecule, and particularly provides a reaction system for detecting the target nucleic acid molecule. The reaction system comprises guide RNA, Cas12b (formerly known as C2c1) and a nucleic acid probe, and the guide RNA, the Cas12b (formerly known as C2c1) and thenucleic acid probe are detected after the reaction is completed. In addition, by combining with the nucleic acid amplification technology (such as LAMP), the sensitivity of the detection method can be greatly improved. The detection system can be used for rapid detection of pathogenic microorganisms, gene mutation, single nucleotide polymorphism, specific target DNA and the like, and can quantifynucleic acid samples.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular, the invention relates to the application of high temperature resistant Cas protein and the detection method and kit of target nucleic acid molecules. Background technique [0002] Rapid nucleic acid molecular detection is a very critical technology in the fields of public health, environmental detection, and criminal investigation. Nucleic acid molecular detection can not only be used to detect whether people, animals and plants are infected with pathogens, but also detect genetic diseases or cancer risk detection, provide auxiliary reference for personal medication, and whether there is microbial pollution in water bodies. At present, many nucleic acid detection methods have been developed, such as Realtime PCR, FISH hybridization technology (Fluorescence in situ hybridization), and the like. However, there is still a need to develop rapid, cheap, and sensitive nucleic acid detection...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2521/101C12Q2521/337C12Q1/6816C12Q1/6827C12N2310/20Y02A50/30C12Q2521/301C12Q2563/107C12Q2565/1015C12N9/22C12N15/113C12Q2527/125C12Q2521/107C12Q1/683C12Q2545/10C12Q2600/158
Inventor 李诗渊王金
Owner SHANGHAI TOLO BIOTECH CO LTD
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