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Chemical derivatization method and application thereof to nucleic acid modification detection by liquid chromatogram-mass spectrometer (LC-MS) method

A derivatization and chemical technology, applied in biochemical equipment and methods, microbial determination/inspection, measurement devices, etc., can solve the problems of insufficient sensitivity to detect, cumbersome operation, time-consuming and labor-intensive, etc., to facilitate quantitative analysis, avoid Contamination mass spectrometry, the effect of broad application prospects

Inactive Publication Date: 2015-04-08
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method of sample pretreatment is cumbersome and time-consuming.
And if the enrichment and purification process of HPLC is not carried out, the sensitivity of LC-MS method may not be enough to detect 5-foC or 5-caC in genomic DNA

Method used

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  • Chemical derivatization method and application thereof to nucleic acid modification detection by liquid chromatogram-mass spectrometer (LC-MS) method
  • Chemical derivatization method and application thereof to nucleic acid modification detection by liquid chromatogram-mass spectrometer (LC-MS) method
  • Chemical derivatization method and application thereof to nucleic acid modification detection by liquid chromatogram-mass spectrometer (LC-MS) method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Human Cell DNA Analysis

[0045] Three human cell lines: HeLa (cervical carcinoma cells), Jurkat-T (leukemic lymphocytes) and 293T (human embryonic kidney cells). Take a certain number of cultured cells and wash them with PBS buffer, put them in a 1.5mL centrifuge tube, centrifuge at 2000g for 20s to pellet the cells, and discard the supernatant. Add 0.5mL cell lysate A (320mM sucrose, 5mM MgCl 2 , 10mM Tris, 0.1mM deferoxamine, pH 7.5, 1% Triton X-100), shake vigorously for 30s with a micro-vortex mixer. The sample was centrifuged at 16000g for 20s, the supernatant was discarded, and the previous process was repeated. Then add 0.2 mL of cell lysate B (10 mM Tris, 5 mM EDTA, 0.15 mM deferoxamine, pH 8.0, 1% sodium lauryl sarcosine), and shake vigorously for 30 s with a micro-vortex mixer. Add 10 μL RNaseA (1 mg / mL) and 3 μL RNaseT1 (1 U / μL), and incubate in a water bath at 50° C. for 15 minutes to remove RNA. Then add 10 μL proteinase K (20mg / ml in H 2 O...

Embodiment 2

[0049] Example 2: DNA Analysis of Cancer Tissues and Paracancerous Tissues of Colorectal Cancer Patients

[0050] Take formalin-fixed, paraffin-embedded colorectal patient tissue sections (5-10 slices, about 30 mg), and use a paraffin-embedded tissue DNA extraction kit ( FFPE DNA Kit, Omega Co.) to extract DNA from tissues. The extracted DNA was dissolved in water and quantified by an ultra-micro ultraviolet spectrophotometer. Take 2-20μg DNA, dry it in vacuum, add 17μL water and 2μL S1 nuclease buffer (300mM sodium acetate, pH 4.6, 2800mM sodium chloride, 10mM zinc sulfate) in sequence, put it in a 95℃ water bath for 5 minutes, then place it quickly Quench in an ice-water bath for 2 minutes to untangle the DNA double strands, add 1 μL of S1 nuclease (180 U / μL) and incubate in a 37°C water bath for 12-16 hours. Then add 65 μL water, 10 μL alkaline phosphatase buffer (500 mM Tris-HCl, 100 mM magnesium chloride, pH 9.0), 1 μL alkaline phosphatase (30 U / μL), 4 μL snake venom p...

Embodiment 3

[0053] Example 3: Yeast Cell DNA Analysis

[0054]Ten yeast strains: Saccharomyces cerevisiae BY4741, Saccharomyces cerevisiae W1588-4C, Debaryomyces hansenii, Schizosaccharomyces pombe, Maxk Kluyveromyces marxianus, Schizosaccharomyces octosporus, Yarrowia lipolytica, Pichia pastoris, Saccharomyces boulardii, Krull lactis After the culture of Kluyveromyces lactis, the yeast cell suspension was centrifuged at 4600g for 5 minutes, the cell pellet was collected and washed with water, and the residual medium was removed. Disperse the yeast cells in 1 mL of cell lysate (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl, 10 g / L SDS, 20 g / L Triton X-100), and then add an equal volume of phenol / chloroform (1: 1. v / v, phenol is saturated with 10mM Tris, 1mM EDTA, pH 8) and 1.5g glass beads (425-600μm), and shake vigorously for 10min with a micro-vortex mixer. Then 1 mL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was added to the solution, and centrifuged at 13500 g for 5 minutes. ...

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Abstract

The invention discloses a chemical derivatization method and an application thereof to nucleic acid modification detection by a liquid chromatogram-mass spectrometer (LC-MS) method. A derivatization strategy is performed on 5-methylcystein, 5-hydroxymethylcytosine, 5-aldehyde cytosine and 5-carboxylcytosine in deoxyribonucleic acid (DNA) by means of 2-bromo-1-(4-dimethylamino-phenyl)-ethyl ketone (BDAPE). Retention behaviors of modified nucleosides in reversed phase liquid chromatography are remarkably improved, and meanwhile, the detection sensitivity of the modified nucleosides in mass spectra is greatly improved. The chemical derivatization method has the advantages that the sensitivity and the selectivity are high, the operation is simple and convenient, quantitative detection of cytosine modification with extremely low abundance in the DNA can be achieved without complicated sample pretreatment, and the method can be applied to analysis of diseases and study on mutual relation among the 5-methylcystein, the 5-hydroxymethylcytosine, the 5-aldehyde cytosine and the 5-carboxylcytosine.

Description

technical field [0001] The invention relates to a method of chemical derivatization combined with LC-MS and its application in DNA and RNA modification analysis. Background technique [0002] In mammalian DNA, the 5-position of cytosine will undergo methylation modification under the action of DNA methyltransferase to form 5-methylcytosine (5-mC). 5-mC participates in many important biological processes, such as genome imprinting, gene expression regulation, etc., and is an important epigenetic modification. 5-methylcytosine can be further oxidized successively by TET proteins to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC) and 5-carboxycytosine (5-caC) , forming a new modification. These newly discovered modifiers are closely related to a variety of important physiological functions, such as cell differentiation, cell reprogramming, nervous system development, the occurrence and development of diseases, and so on. The DNA modification level of normal people ...

Claims

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Application Information

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IPC IPC(8): G01N30/06G01N30/02C12Q1/68
Inventor 袁必锋冯钰锜唐阳
Owner WUHAN UNIV
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