Detection method of oxidation modified nitrogenous bases

A detection method, nitrogen base technology, applied in the field of DNA modification detection, can solve the problem of indetermination of 8-oxoG modification damage, etc., and achieve the effect of convenient detection and precise treatment

Inactive Publication Date: 2018-12-28
QINGDAO UNIV
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Problems solved by technology

These methods all have the characteristics of high detection sensitivity, but they measure the amount of all oxidation products in DNA or the total content of 8-oxoG in tissues, and cannot determine the extent of damage caused by 8-oxoG modification in genomic DNA. Specific site

Method used

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  • Detection method of oxidation modified nitrogenous bases
  • Detection method of oxidation modified nitrogenous bases
  • Detection method of oxidation modified nitrogenous bases

Examples

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Effect test

Embodiment 1

[0040] The method for positioning and detecting base (G) oxidation modification in a DNA sequence based on SMRT sequencing technology, specifically comprises the following steps:

[0041] 1) Using a 155bp long DNA sequence produced by Takara Clontech Company with two 8-oxoG sites, two oxidatively modified guanines are located at the 34th and 57th positions in the upstream of the DNA sequence, and the DNA oxidation modification product 8-hydroxyl The chemical structure of deoxyguanine is as figure 1 shown;

[0042] 2) The sequence sample obtained in step 1) is constructed into a gene library;

[0043] 3) The library of step 2) is sequenced on the PacBio RSII sequencer to obtain the original sequence;

[0044]4) Step 3) The original sequence uses SMRT Link v5.1.0 software for data quality control and filtering, and then the sequence is exported for bioinformatics analysis;

[0045] 5) Use a known synthetic sequence as a reference sequence, step 4) sequence and reference sequ...

Embodiment 2

[0052] The method for identifying the oxidative modification site of plasmid DNA pcDNA3.1 8-oxoG by using SMRT sequencing technology and base modification site analysis method specifically includes the following steps:

[0053] 1) Extract plasmid pcDNA3.1 (5477bp), using Tiangen High Purity Plasmid Extraction Kit (non-centrifugal column type, DP116). The specific method is as follows:

[0054] a) Take 100 mL of overnight cultured bacterial solution, and centrifuge at room temperature at 10,000 rpm for 3 minutes to collect the bacteria.

[0055] b) Aspirate the supernatant as much as possible, add 10mL solution P1 to the centrifuge tube with the cell pellet left, and vortex to thoroughly suspend the cell pellet.

[0056] c) Add 10 mL of solution P2 to the centrifuge tube, immediately turn it up and down gently 6-8 times to fully lyse the bacteria, and place it at room temperature for 5 minutes.

[0057] d) Add 10mL solution P4 to the centrifuge tube, immediately turn it gentl...

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Abstract

The invention relates to the field of DNA modification detection, in particular to a detection method of oxidation modified nitrogenous bases. IPD (interpulse duration) values of different nitrogenousbases are measured with the detection method of oxidation modified nitrogenous bases, and the oxidation modified nitrogenous bases are obtained according to different IPD values. The IPD values of the oxidation modified nitrogenous bases are found to be different from those of unmodified nitrogenous bases, and accordingly, whether the nitrogenous bases are modified can be distinguished.

Description

technical field [0001] The invention relates to the field of DNA modification detection, in particular to a method for detecting oxidatively modified nitrogenous bases. Background technique [0002] DNA stores the genetic information that organisms rely on for survival and reproduction. When affected by external environment and internal factors of organisms, DNA molecules will be damaged or changed. Free radicals are a class of highly active substances that can directly or indirectly play a strong oxidizing role. Common forms of reactive oxygen species (ROS) include hydroxyl radicals (OH), superoxide anions (O 2- ) and hydrogen peroxide (H 2 o 2 ), etc., which contain active single electrons, which are very easy to combine with nucleophilic DNA molecules, resulting in the modification of DNA bases. Among them, the oxidation of the C8 position of guanine (forming 8-hydroxydeoxyguanine, 8-oxoG) is one of the most common DNA oxidative modifications triggered by reactive oxy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6886C12Q1/6869
CPCC12Q1/6869C12Q1/6883C12Q1/6886C12Q2537/165
Inventor 李培峰曾焕玉高金宁于忠杰
Owner QINGDAO UNIV
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