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Buffer solution for cas12a editing DNA as well as preparation method and application of buffer solution

A buffer and editing technology, which is applied in the direction of recombinant DNA technology, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems that cannot meet the experimental requirements, and achieve the effect of convenient operation, simple preparation process and high practicability

Pending Publication Date: 2021-11-05
天益健康科学研究院(镇江)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the sensitivity of cas12a cutting influenza B DNA with general reaction buffer is only 10pm, which cannot meet the experimental requirements

Method used

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  • Buffer solution for cas12a editing DNA as well as preparation method and application of buffer solution
  • Buffer solution for cas12a editing DNA as well as preparation method and application of buffer solution
  • Buffer solution for cas12a editing DNA as well as preparation method and application of buffer solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The general buffer ratio is as follows:

[0032] components concentration Tris-HCl 40mM NaCl 60mM Mgcl 2

6mM

[0033] Preparation:

[0034] Prepare the Tris-HCl solution with the required concentration, adjust the pH to 7.5, then add KCl and MgCl2 in sequence according to the required concentration, and mix well to obtain the product.

[0035] The method for detecting influenza B DNA by cas12a using the above buffer solution is as follows:

[0036] 1) After the buffer solution is prepared, take a certain ratio and mix it with cas12a, crRNA, B-flow DNA, DNA reporter and RNase inhibitor at various dilution concentrations, and react at 37°C for 30 minutes. The concentration of B stream DNA is 10pM, 1pM, 100fM, 10fM, 0, corresponding to 10 7 、10 6 、10 5 、10 4 , 0 copy number / microliter;

[0037] 2) Put the mixed reaction solution in a tube rack at room temperature, add 30ul of pure water to mix, then put a disposable nucleic a...

Embodiment 2

[0041] The improved buffer ratio is as follows:

[0042]

[0043]

[0044] Preparation:

[0045] Prepare the HEPES solution with the required concentration, adjust the pH to 7.5, then add KCl, MgCl2, glycerin and DTT in sequence according to the required concentration, and mix well to obtain the product.

[0046] The method for detecting influenza B DNA by cas12a using the above buffer solution is as follows:

[0047] 1) After the buffer solution is prepared, take a certain ratio and mix it with cas12a, crRNA, B-flow DNA, DNA reporter and RNase inhibitor at various dilution concentrations, and react at 37°C for 30 minutes. The concentration of B stream DNA is 10pM, 1pM, 100fM, 10fM, 0, corresponding to 10 7 、10 6 、10 5 、10 4 , 0 copy number / microliter;

[0048] 2) Put the mixed reaction solution in a tube rack at room temperature, add 30ul of pure water to mix, then put a disposable nucleic acid detection test strip into each reaction tube, and wait for the reactio...

Embodiment 3

[0052] The improved buffer ratio is as follows:

[0053] components concentration HEPES 18mM Kcl 150mM Mgcl 2

12mM glycerin 0.8％ DTT 0.5mM

[0054] Preparation:

[0055] Prepare the HEPES solution with the required concentration, adjust the pH to 7.5, then add KCl, MgCl2, glycerin and DTT in sequence according to the required concentration, and mix well to obtain the product.

[0056] The method for detecting influenza B DNA by cas12a using the above buffer solution is as follows:

[0057] 1) After the buffer solution is prepared, take a certain ratio and mix it with cas12a, crRNA, B-flow DNA, DNA reporter and RNase inhibitor at various dilution concentrations, and react at 37°C for 30 minutes. The concentration of B stream DNA is 10pM, 1pM, 100fM, 10fM, 0, corresponding to 10 7 、10 6 、10 5 、10 4 , 0 copy number / microliter;

[0058] 2) Put the mixed reaction solution in a tube rack at room temperature, add 30ul of pu...

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Abstract

The invention discloses a buffer solution for cas12a editing DNA as well as a preparation method and application of the buffer solution. The buffer solution is prepared from the following raw materials: 5 to 25 mM of HEPES with the pH of 6.8 to 7.7, 80 to 300 mM of KCl, 5 to 20 mM of MgCl2, 0.5 to 2 percent of glycerol and 0.3 to 0.7 mM of DTT. The preparation method comprises the following steps: preparing an HEPES solution with the required concentration, adjusting the pH value, sequentially adding KCl, MgCl2, glycerol and DTT according to the required concentration, and uniformly mixing to obtain the buffer solution. The HEPES in the invention belongs to a high-purity biological buffering agent, and does not cause degradation and other influences on all reaction components; the buffer solution provided by the invention can increase the sensitivity of cas12a for detecting influenza B DNA by 10-100 times, and the highest sensitivity can reach 100 fM; and the preparation technology is simple, operation is convenient, and high practicability is achieved.

Description

technical field [0001] The invention belongs to the technical field of DNA editing buffer, and in particular relates to a buffer for cas12a editing DNA, a preparation method and application thereof. Background technique [0002] So far, 6 types and more than 20 subtypes of CRISPR-Cas systems have been discovered. Among them, CRISPR-Cas12a is the second type (type V) CRISPR-Cas system for editing mammalian genomes. Cas12a can cut DNA and can complement the CRISPR-Cas9 system. The Cas12a (Cpf1) protein has a RuvC endonuclease domain similar to Cas9. Cpf1 does not need tracrRNA, only crRNA, which is very beneficial to genome editing; Cas12a is smaller than Cas9, and has smaller crRNA molecules (nearly half of the total sgRNA of Cas9); Cas12a-crRNA complex recognizes T-rich PAM bases sequence to cleave target DNA, in contrast to Cas9, which requires G-rich PAM. [0003] First, the Cas12a / crRNA binary complex recognizes the target DNA sequence, forms a ternary complex, and st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2521/327
Inventor 李超姚颜萍严园园张少伦汪智婷
Owner 天益健康科学研究院(镇江)有限公司
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