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Buffer solution for enhancing activity of TET enzyme

A technology of buffer solution and reaction buffer solution, applied in the field of biology

Pending Publication Date: 2022-01-28
翌圣生物科技(上海)股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many TET protein variants on the market for DNA methylation detection, but there is little research on TET enzyme buffer. Therefore, a high-performance TET enzyme buffer is developed to improve the oxidation ability of TET enzyme. The development of in vitro DNA methylation technology and its application in the field of disease diagnosis are of great value

Method used

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  • Buffer solution for enhancing activity of TET enzyme
  • Buffer solution for enhancing activity of TET enzyme
  • Buffer solution for enhancing activity of TET enzyme

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Effects of four buffer systems on TET enzyme activity.

[0028] In this example, we verified the 3-(N-morpholino)propanesulfonic acid sodium salt-sodium hydroxide buffer system (Mops-NaOH), tris-hydrochloric acid buffer system (Tris-HCl) , bis-hydroxyethyltrismethylolmethylamine-sodium hydroxide buffer system (Bis-Tris-NaOH) or 4-hydroxyethylpiperazine sulfuric acid-potassium hydroxide buffer system (Hepes-KOH) four reactions The impact of the system on each TET enzyme, the specific implementation is as follows:

[0029] TET enzyme reaction buffer includes 10-100 mM buffer system (pH 7.5), 10-300 mM sodium chloride, 0.1-10 mM ascorbic acid (L-AA), 0.1-10 mM s adenosine triphosphate (ATP), 0.1-10 mM α - Ketoglutarate (α-KG), 30-300 uM ferrous ammonium sulfate, 0.1-5 mM dithiothreitol.

[0030] Epigentek's Epigenase 5mC-hydroxylase TET activity / inhibition assay kit (fluorescence method) was used to measure NgTET1, mTET1, mTET2, hTET1, hTET2, and hTET3 accordi...

Embodiment 2

[0031] Example 2: Effect of small molecule additives on TET enzyme activity.

[0032] In this example, we verified the effects of different small molecule additives on TET enzyme activity. Small molecule additives are 1 mM hydroquinone, 1 mM tetrafluoro-p-benzoquinone, 1 mM tetrachloro-p-benzoquinone, 1 mM tetrabromo-p-benzoquinone, 1 mM tetraiodobenzoquinone, 1 mM L-cysteine , 1 mM L-proline, 1 mM potassium L-glutamate, 1 mM tris(2-carboxyethyl)phosphine, 1 mM dithiothreitol, 1 mM thioethylene glycol. Using Epigentek's Epigenase 5mC-hydroxylase TET activity / inhibition assay kit (fluorescence method), add the above-mentioned small molecule additives respectively, and measure the enzyme activity of TET according to the procedure in the manual. The enzyme activity assay results were as figure 2 , phenol and quinone compounds (hydroquinone, tetrafluoro-p-benzoquinone, tetrachloro-p-benzoquinone, tetrabromo-p-benzoquinone and tetraiodobenzoquinone), amino acid analogs (L-cystei...

Embodiment 3

[0033] Example 3: Effects of concentrations of hydroquinone, chloro-p-benzoquinone, L-cysteine ​​and tris(2-carboxyethyl)phosphine on TET enzyme activity.

[0034] In this example, we verified the effects of hydroquinone, chloro-p-benzoquinone, L-cysteine ​​and tris (2-carboxyethyl) phosphine on the enzyme activity of NgTET1 in the TET enzyme reaction buffer. Refer to Examples 1 and 2 for the flow process. The result is as image 3 As indicated, 1-5 mM hydroquinone, 0.5-1 mM chloranil, 2-10 mM L-cysteine, and 0.1-0.5 mM tris(2-carboxyethyl)phosphine have the effect on TETase Facilitation works best.

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Abstract

The invention provides a reaction buffer solution for enhancing activity of the an enzyme. The solution comprises 3-(N-morpholinyl) propanesulfonic acid sodium salt, bis (2-ethoxyl) amino-tris (hydroxymethyl) methane, hydroxyethyl piperazine ethanesulfonic acid, sodium chloride, ascorbic acid, citric acid, alpha-ketoglutaric acid, 1,3-acetone dicarboxylic acid, adenosine triphosphate, hydroquinone, tetrafluoro-p-benzoquinone, tetrachloro-p-benzoquinone, tetrabromo-p-benzoquinone, tetraiodobenzoquinone, L-cysteine, L-proline, L-potassium glutamate, tris (2-carboxyethyl) phosphine, thioglycol, ferrous sulfate, ethylenediamine ferrous sulfate, ammonium ferrous sulfate, ferrous citrate, ammonium ferrous citrate, ferrous chloride, 2, 6-bis (1, 1-bis (2-pyridine) ethyl) pyridine-iron oxide complex and the like. The oxidation activity of the TET enzyme on a 5mC substrate can be remarkably enhanced, the sensitivity and accuracy of a DNA methylation detection technology based on TET enzyme oxidation reaction can be improved. The buffer solution can be applied to disease diagnosis, especially the field of tumor early screening.

Description

technical field [0001] The patent of the invention relates to a buffer component for enhancing TET enzyme activity, and belongs to the field of biotechnology. Background technique [0002] DNA cytosine methylation (5mC) is the most common base modification in DNA, accounting for about 1%-8% of all cytosines, and is called the "fifth base". DNA methylation is significantly correlated with chromatin state and gene transcription activity, and is an effective basis for predicting gene expression levels. Therefore, the detection of DNA methylation level is an effective means for clinical disease diagnosis. The existing DNA methylation detection technology mainly relies on the bisulfite conversion method of reverse screening. The principle is to use bisulfite to convert unmethylated cytosine into uracil, and then amplify it by PCR. into thymine. This method suffers from large DNA damage, high background noise, and low accuracy. In recent years, the enzymatic conversion methyla...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2600/154C12Q2527/125C12Q2527/127C12Q2521/50
Inventor 侯策韦磊江翱陈晶晶黄开喻滕以刚曹振宋东亮
Owner 翌圣生物科技(上海)股份有限公司