Buffers and their applications

A technology of buffer and acetate buffer, which is applied in the field of joint connection buffer, buffer, and construction of nucleic acid library, which can solve the limitations of high-throughput workflow construction, limited height of the connection reaction plateau, and inability to advance the connection time, etc. problems, to achieve the effect of improving the efficiency of the ligation reaction, reducing the amount of enzyme used in the system, and having a small inhibitory effect

Active Publication Date: 2021-04-06
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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  • Buffers and their applications
  • Buffers and their applications
  • Buffers and their applications

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 general steps, wherein the composition of the ligation buffer was 45 mM Tris-AC buffer, 3 mM Mg 2+ 、Na + , 5mM dithiothreitol (DTT), 10% PEG4000-8000 and 1.5mM ATP, the pH value is 8. Wherein, the Na concentration in the connection buffer is as shown in Table 1, specifically as follows:

[0076] Table 1 Statistics of the number of linkers self-ligating 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. With the increase of Na+ concentration, the number of adapter self-associations decreased significantly, and the number of adapter self-associations decreased significantly in the presence of 37.5mM Na+. The number is about 4 times that of 50mM Na+, and the number of adapter self-ligations in the presence...

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 buffering capabilities of these two types are also different. Furthermore, in this example, T4 DNA connection is compared 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, the pH value is 8. Only for the Tris buffer in the connection buffer are Tris-AC and Tris-HCl respectively, the buffering effect of the buffer is as follows image 3 As shown, in the presence of 7.5% (w / v) PEG, Tris-AC has a wider fluctuation range, but has a positive effect on the increase of library yield.

Embodiment 3

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

[0085] Table 2 Library Yield Statistics at Different PEG Concentrations

[0086]

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

[0088] Wherein, it should be noted that 15 min refers to the ligation time of 15 min, and 15 min* means that the ligation r...

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Abstract

The invention discloses a buffer and an application thereof, wherein the buffer comprises: 33-66mM Tris buffer, 1.6-5mM divalent cations, 25-75mM monovalent cations, 0.5-10mM dithiothreitol (DTT), 5‑15% PEG4000‑8000 and 0.5‑2mM ATP, pH 7‑9. The buffer can improve the efficiency of the ligation reaction, increase the conversion rate of the substrate, reduce the mismatch rate of the ligation reaction, and has good compatibility and little inhibitory effect on the ligase. At the same time, it can significantly reduce the amount of enzyme used in the system, thereby reducing the experimental the cost of.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a buffer and its application, more specifically to a buffer, a kit, the buffer used in the construction of a nucleic acid library as an adapter ligation buffer, and a Methods for constructing nucleic acid libraries. Background technique [0002] The general process for constructing a next-generation sequencing library includes: (1) Fragmenting the target DNA; (2) Blunting the free DNA; (3) Protruding adenylation at the 3' end of the blunt DNA ; (2) Ligate the protruding adenylated DNA fragment with the protruding thymine-prominent double-stranded Y-shaped linker. The purpose of library construction is to add adapters to the two segments of double-stranded DNA fragments. The library conversion rate is the ratio of the measured yield to the theoretical maximum yield quality check, that is, how many initial samples are finally converted into two fragments with adapters. [00...

Claims

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Application Information

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Patent Type & Authority Patents(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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