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Buffer solution and application thereof

A buffer and acetate buffer technology, which is used in the field of linker ligation buffer, buffer, and nucleic acid library construction, can solve the problem of the limited height of the ligation reaction platform, the limitation of high-throughput workflow construction, and the inability to advance the connection time. To improve the efficiency of the ligation reaction, reduce the amount of enzyme used in the system, and achieve a small inhibitory effect

Active Publication Date: 2020-05-05
ZHEJIANG ANNOROAD BIO TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The conversion rate is largely affected by the ligation efficiency. The ligation reaction efficiency is related to many factors such as the enzyme reaction efficiency, the openness of the DNA chain end, the collision efficiency of the donor and the acceptor, and the linker in the ligation reaction system will also The effective number of adapters generated in the self-ligating depletion reaction is an efficiency bottleneck in the library construction process
At present, the common rapid connection buffer in the market has a limited connection reaction plateau height, and there are situations such as increasing the amount of enzyme input to overcome the mismatch of the buffer system, which increases the cost of practical application; in addition, the connection time under the existing connection buffer Unable to push forward the enzyme reaction balance; and a large number of mismatches will be generated during the premixing process, which reduces the library yield and limits the construction of high-throughput workflows
[0004] Therefore, the existing ligation buffer needs to be improved

Method used

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  • Buffer solution and application thereof
  • Buffer solution and application thereof
  • Buffer solution and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] In this example, the effect of different concentrations of monovalent cations on the yield of the library in the presence of PEG was studied according to the above-mentioned general steps. Among them, the composition of the ligation buffer is 45mM Tris-AC buffer, 3mM Mg 2+ , Na + , 5mM dithiothreitol (DTT), 10% PEG4000-8000 and 1.5mM ATP, pH value is 8. Among them, the Na+ concentration in the connection buffer is shown in Table 1, and the details are as follows:

[0076] Table 1 Statistics of the number of self-connections of the joints under different Na+ concentrations

[0077]

[0078] As shown in Table 1, in the presence of 7.5% (w / v) PEG, the results are in line with the hypothesis. As the concentration of Na+ increases, the number of linkers self-ligates significantly. In the presence of 37.5mM Na+, the number of linkers self-ligates The amount is about 4 times that of 50mM Na+, and the number of self-linking linkers in the presence of 37.5mM Na+ is about 6.5 times th...

Embodiment 2

[0082] Since the negative ions of Tris-AC and Tris-HCl have different electronegativity, they have different effects on the interaction between enzymes and DNA, and the two buffering capabilities are also different. Furthermore, in this example, compare T4 DNA connection In the enzyme-mediated ligation reaction, the effect of Tris-AC and Tris-HCl buffer is carried out according to the above general steps. The composition of the ligation buffer is Tris-AC buffer, 3mM Mg 2+ , 50mM Na + , 5mM dithiothreitol (DTT), 10% PEG4000-8000 and 1.5mM ATP, pH value is 8. Only the Tris buffer in the connection buffer is Tris-AC and Tris-HCl. The buffer effect of the buffer is as follows image 3 As shown, in the presence of 7.5% (w / v) PEG, Tris-AC has a wide fluctuation range, but it has a positive effect on the increase in library yield.

Embodiment 3

[0084] In this example, according to the general method of constructing a nucleic acid library, the concentration of PEG in the ligation buffer was changed to compare the effects of 7.5% and 10% PEG on library construction. The composition of the ligation buffer is 45mM Tris-AC buffer, 3mMMg 2+ , 50mM Na + , 5mM dithiothreitol (DTT), PEG4000-8000 and 1.5mM ATP, pH value of 8, as follows:

[0085] Table 2 Library yield statistics under different PEG concentrations

[0086]

[0087] As shown in Table 2, when the concentration of PEG6000 is 10% and the connection time is 15 minutes, the library yield is 1.62 times that of the 7.5% concentration. When the premix was prepared and placed for 1 hour, the yield of the library under the condition of 10% PEG6000 was still higher than that of the concentration of 7.5% PEG6000.

[0088] Among them, it should be noted that 15min means that the ligation time is 15min, and 15min* means that the ligation reaction reagent mixture (except the substra...

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Abstract

The invention discloses a buffer solution and application thereof. The buffer solution is prepared from a Tris buffer solution with a concentration of 33 to 66 mM, divalent cations with a concentration of 1.6 to 5 mM, monovalent cations with a concentration of 25 to 75 mM, dithiothreitol (DTT) with a concentration of 0.5 to 10 mM, PEG 4000 to 8000 with a concentration of 5 to 15% and ATP with a concentration of 0.5 to 2 mM. The pH value of the Tris buffer solution is 7 to 9. The buffer solution can improve the efficiency of a ligation reaction, increase the conversion rate of a substrate and reduce the mismatch rate of the ligation reaction, is good in compatibility and small in inhibition effect on ligase, and remarkably reduces the enzyme consumption of a system, so experiment cost is reduced.

Description

Technical field [0001] The present invention relates to the field of biotechnology, in particular, to buffers and their applications, and more specifically to a buffer, a kit, the buffer used in the construction of a nucleic acid library as a linker ligation buffer, and a Methods of constructing nucleic acid libraries. Background technique [0002] The general process of constructing a second-generation sequencing library includes: (1) Fragmentation of the target DNA; (2) End-blending treatment of free DNA; (3) Protruding adenylation at the 3'end of the blunted DNA (2) Connect the overhanging adenylated DNA fragment with the overhanging thymidine double-stranded Y-linker. The purpose of library construction is to add linkers to the two double-stranded DNA fragments. The library conversion rate is the ratio of the measured yield to the theoretical maximum yield quality inspection, that is, how many starting samples are finally converted into two fragments with adapters. [0003] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806
CPCC12Q1/6806C12Q2527/125C12Q2525/191C12Q2521/501
Inventor 潘伟业王亚蕾程世月李志民李大为玄兆伶王海良王娟
Owner ZHEJIANG ANNOROAD BIO TECH CO LTD
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