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Method and kit for marking nucleic acid in living cell

A technology of living cells and kits, which is applied in the field of a method and kits for labeling nucleic acids in living cells, can solve problems such as difficulty in repeating and unstable experimental results, and achieve fast response time, mild conditions, and detection of cell growth. Effect

Active Publication Date: 2010-12-22
广州锐博生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to detect the probe inserted into the nucleic acid molecular chain, it is necessary to denature the nucleic acid molecule through various conditions. The conditions that are often used now include methanol solution of acetic acid, concentrated hydrochloric acid, etc. These denaturing conditions make the experimental results unstable and often produce unrepeatable results.

Method used

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  • Method and kit for marking nucleic acid in living cell
  • Method and kit for marking nucleic acid in living cell
  • Method and kit for marking nucleic acid in living cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Construction of nucleic acid labeling kit

[0047] 1. Nucleoside Analog Synthesis

[0048] 1.1 Synthesis of nucleoside analogs containing dipolar groups

[0049] The present invention has synthesized the dipolar-philic group nucleoside analogs of the structure in (2) (3), 5-ethynyl-2'-deoxyuridine (YTU), 5-ethynyluridine (YU), 2'-deoxy-5-(1,7 octadiyne)-uridine (XTU), 5-(1,7 octadiyne)-uridine (XU), 5-ethyne Base-2'-deoxycytidine (YTC), 5-ethynylcytidine (YC), 2'-deoxy-5-(1,7 octadiyne)-cytidine (XTC), 5-(1,7 octadiynyl)-cytidine (XC), 2'-deoxy-8-ethynyl-adenosine (YTA), 8-ethynyl-adenosine (YA), 2'-Deoxy-8-(1,7 octadiynyl)-adenosine (XTA), 8-(1,7 octadiynyl)-adenosine (XA), 7-ethynyl-7- Aza-2'deoxy-adenosine (QYTA), 7-ethynyl-7-aza-adenosine (QYA), 7-ethynyl-7-aza-2'deoxy-adenosine Glycoside (QYTA), 7-ethynyl-7-aza-adenosine (QYA), 7-(1,7 octadiynyl)-7-aza-2'deoxy-adenosine (QXTA) , 7-(1,7 octadiynyl)-7-aza-adenosine (QXA), 2'-deoxy-8-ethynyl-guanosine...

Embodiment 2

[0087] Example 2: Labeling intracellular DNA and RNA at the cellular level using a "click" reaction kit

[0088] (1) The nucleic acid labeling kit of this embodiment includes:

[0089] Dipolar group nucleoside analogs: 5-ethynyl-2'-deoxyuridine (YTU), 5-ethynyluridine (YU), 2'-deoxy-5-(1,7 Octadiynyl)-uridine (XTU), 5-(1,7 Octadiynyl)-uridine (XU), 5-ethynyl-2'-deoxycytidine (YTC), 5 -ethynylcytidine (YC), 2'-deoxy-5-(1,7 octadiyne)-cytidine (XTC), 5-(1,7 octadiyne)-cytidine (XC), 2'-deoxy-8-ethynyl-adenosine (YTA), 8-ethynyl-adenosine (YA), 2'-deoxy-8-(1,7 octadiyne) - adenosine (XTA), 8-(1,7 octadiyne)-adenosine (XA), 7-ethynyl-7-aza-2'deoxy-adenosine (QYTA), 7-ethynyl-7-aza-adenosine (QYA), 7-ethynyl-7-aza-2'deoxy-adenosine (QYTA), 7-ethynyl-7-aza- Adenosine (QYA), 7-(1,7-octadiynyl)-7-aza-2'deoxy-adenosine (QXTA), 7-(1,7-octadiynyl)-7-aza Hetero-adenosine (QXA), 2'-deoxy-8-ethynyl-guanosine (YTG, 8-ethynyl-guanosine (YG), 2'-deoxy-8-(1, 7-octadiynyl)-guanosine (XTG),...

Embodiment 3

[0096] Example 3: Using different concentrations of copper ions to catalyze the "click" reaction at the cellular level

[0097] (1) The nucleic acid labeling kit of this embodiment includes:

[0098] Nucleoside analogs: YTU

[0099] Biomolecular probes: cyanine azido dyes (1,1,1',1'-tetramethyl-3-ethyl-3'-azidopropyl-indoletrimethylcyanine or 1-ethyl Base-1'-azido-n-hexyl-3,3,3',3'-tetramethyl-6-sulfonic acid group-indole pentamethine cyanine);

[0100] Catalyst: CuSO 4 , sodium ascorbate;

[0101] Solvent: mixed solution of water and DMSO.

[0102] (2) Labeled nucleic acid: Hela cells were seeded in a 96-well plate and cultured with DMEM medium containing 10% fetal bovine serum. After culturing for 24 hours, 5-ethynyl-2'-deoxyuridine (YTU) was added to the culture medium at a final concentration of 1 nM-1000 nM, and cultured for another 24 hours. Remove the culture medium, add 100 μl 4% paraformaldehyde to each well for fixation for 30 minutes, wash with 2 mg / ml glycine...

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Abstract

The invention discloses a method and a kit for marking nucleic acid in a living cell. The kit mainly comprises a nucleoside analogue and a biological molecular probe, wherein the nucleoside analogue can be inserted into a nucleotide sequence and comprises a 1,3-dualbipolar group or dipolephilic group; the biological molecular probe comprises the dipolephilic group or 1,3-dualbipolar group corresponding to the1,3-dualbipolar group or the dipolephilic group; and click reaction can be performed between the 1,3-dualbipolar group and the corresponding dipolephilic group. During marking, the nucleic acid modified by the nucleotide analogue comprising the 1,3-dualbipolar group and the dipolephilic group can be covalently linked with the corresponding active probe molecules through the click reaction. The method and the kit for marking the nucleic acid in the living cell can be further applied to detecting cell proliferation and cell apoptosis, marking a plasmid vector, virus, low-interference RNA and microRNA and researching early development after the nucleic acid is marked.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method and a kit for labeling nucleic acid in living cells. technical background [0002] Cells are the basic unit of life, and the specificity of cells determines the specificity of individuals. Therefore, in-depth research on cells is the key to uncovering the mysteries of life, transforming life and conquering diseases. The main life activities of cells include proliferation, differentiation, senescence and apoptosis, etc. These life activities of cells are of great significance to the survival and development of organisms. Cell proliferation is the basis of organism growth, development, reproduction and inheritance; cell differentiation is an important stage of individual development, the differentiation of germ layer cells leads to tissue formation, organogenesis and system building, and abnormal differentiation of cells can lead to cancer; cell ag...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 张必良王玮渠德忠
Owner 广州锐博生物技术有限公司