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Method for detecting guanines in non double-chain region in nucleic acid

A detection method, the technology of guanine, which is applied in the field of DNA sequencing, can solve the problems of being easily oxidized, and achieve the effect of sensitive detection and effective detection

Inactive Publication Date: 2015-05-20
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although DNA is not easily hydrolyzed, it is easily oxidized

Method used

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  • Method for detecting guanines in non double-chain region in nucleic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Embodiment 1: DNA reacts directly with piperidine

[0021] Take 2 μL of an aqueous solution (10 μM) of fluorescently labeled DNA and add it to a piperidine aqueous solution with a volume concentration of 10%. After heat treatment at 90°C for 0.5 hours, spin the solvent, and load the spin-dried sample into the electrophoresis tank After 2 hours, observe the gel results, no DNA fragmentation occurs.

Embodiment 2

[0022] Example 2: DNA is first treated with potassium tungstate, and then reacted with piperidine

[0023] Take 2 μL of an aqueous solution (10 μM) of fluorescently labeled DNA and treat it with 0.05-5 mM potassium tungstate aqueous solution for 1-10 min, then add ice ethanol to precipitate the DNA, and then centrifuge to remove the supernatant containing potassium tungstate. After the precipitate was spin-dried, it was added to a piperidine aqueous solution with a volume concentration of 10%. After heat treatment at 90°C for 0.5 hours, the solvent was spin-dried; the sample obtained by spin-drying was loaded to the electrophoresis tank, and the gel results were observed after 2 hours. There was no DNA Fracture occurs.

Embodiment 3

[0024] Example 3: DNA is first treated with hydrogen oxide, and then reacted with piperidine

[0025] Take 2 μL of an aqueous solution (10 μM) of fluorescently labeled DNA and treat it with 0.05-5 mM hydrogen peroxide solution for 1-10 min, then add ice ethanol to precipitate the DNA, and then centrifuge to remove the supernatant containing hydrogen peroxide. After the precipitate was spin-dried, it was added to a piperidine aqueous solution with a volume concentration of 10%. After heat treatment at 90°C for 0.5 hours, the solvent was spin-dried; the sample obtained by spin-drying was loaded to the electrophoresis tank, and the gel results were observed after 2 hours. There was no DNA Fracture occurs.

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Abstract

The invention discloses a new method for detecting guanines in a non double-chain region in DNA by using a mixed aqueous solution of hydrogen peroxide and potassium tungstate. The method comprises the following steps: enabling hydrogen peroxide and potassium tungstate to selectively have oxidization reaction with guanine in DNA and then treating with piperidine so that oxidized DNA is fractured; and carrying out polyacrylamide gel electrophoresis experiment so that nucleic acid fracture zones with different thicknesses are separated one by one, thus obtaining the positions and number of all guanines. The method can be used for quickly, effectively and sensitively detecting guanines in the non double-chain region.

Description

technical field [0001] The invention relates to a new method for detecting guanine in a non-double-stranded region in nucleic acid by using a mixed aqueous solution containing potassium tungstate and hydrogen peroxide, and belongs to the field of DNA sequencing. Background technique [0002] DNA cleavage and damage are important factors in cellular processes. In addition to natural protein nucleases that degrade DNA, several different chemical nucleases have been identified that cleave DNA, primarily through hydrolysis and oxidation. We know that in addition to single-stranded and double-stranded DNA, there are many non-classical non-double-stranded structures, such as hairpin structures, protrusions, loop structures, nodules, etc., which are generally believed to exist in double-stranded DNA. Double-stranded structures are errors generated during DNA replication, and these structures play an important role in chromosomal mutations in cancer cells, so it is possible to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 周翔毛伍祥宋鸿玮翁小成
Owner WUHAN UNIV
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