Method for screening disease marker

A technology for markers and diseases, applied in biochemical equipment and methods, library screening, microbial determination/testing, etc., to achieve the effect of overcoming difficult separation and broad application prospects

Inactive Publication Date: 2009-09-23
谭蔚泓
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, for disease marker molecules on the surface of non-protein cell membranes, the above-mentioned proteomics methods are powerless

Method used

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  • Method for screening disease marker
  • Method for screening disease marker

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Experimental program
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Embodiment 1

[0029] Example 1. Preparation of molecular probes related to leukemia

[0030] (1) Screening of nucleic acid aptamers related to leukemia

[0031] 1. Synthesis of random nucleic acid library

[0032] According to the literature (Jayasena S D. Aptamers: an emerging class of molecules thatrival antibodies in diagnostics. Clin. Chem. 1999, 45: 1628-1650), the following random nucleic acid library was designed and synthesized: 5'-ATA CCA GCT TAT TCA ATT-52-nt-AGA TAG TAAGTG CAATCT-3'.

[0033] 2. Screening of nucleic acid aptamers related to leukemia

[0034] Binding buffer: containing 4.5g / L glucose, 5mM MgCl 2 , 1mM CaCl 2 , 2.67mM KCl, 1.47mM potassium dihydrogen phosphate, 137.93mM sodium chloride, 8.06mM disodium hydrogen phosphate, 0.1mg / mL tRNA (R8759 Sigma, Ribonucleic acid, transfer from baker's yeast (S.cerevisiae)) and 1mg / mL Bovine serum albumin (Fisher), pH 7.4.

[0035] CCRF-CEM cells (CCL-119) and Ramos cells (CRL-1596) were purchased from ATCC (American Type ...

Embodiment 2

[0059] Example 2. Preparation of leukemia markers using leukemia-associated nucleic acid aptamers

[0060] 1. Preparation of markers

[0061] The nucleic acid aptamer molecule sc1 is labeled with biotin to make a molecular probe sc1-pro-bio.

[0062] Take 10×10 8 CCEM cells were lysed on ice with 50mM Tris-hydrochloric acid buffer for 20 minutes, centrifuged to remove the supernatant, and the pellet was lysed with phosphate buffer containing 0.3% Triton-100 for 1 hour on ice, and the supernatant was obtained by centrifugation.

[0063] After the supernatant was incubated with 1 nmol of molecular probe sc1-pro-bio on ice for 30 minutes, 200 μL of streptavidin-labeled magnetic particles (1 micron, Dynal Biotech ASA, Oslo, Norway) were added and incubated for 5 minutes, Adsorb the magnetic particles with a magnet, remove the supernatant; wash the magnetic particles three times with phosphate buffer, add the magnetic particles to 30 μL of polyacrylamide gel electrophoresis (PAGE...

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Abstract

The invention discloses a method for screening disease markers. The method comprises the steps as follows: (a) a nucleic acid library is screened by target disease cell in vitro to obtain aptamer related to the target disease; the nucleic acid library is library of single stranded random nucleotide sequence; (b) the aptamer related to the disease is combined with the target molecule on the target disease cell in vitro to obtain the compound of the aptamer and the target molecule, then separating the compound to obtain the target molecule on the target disease cell that is the disease marker. Compared with the prior art, the method for preparing the disease markers in the invention is suitable for separating the membrane protein markers and also suitable for separating the non-membrane protein markers, and overcomes the defect that the proteome method in the prior art is difficult to separate the membrane protein markers; furthermore, the method of the invention can separate the disease marker molecules on surface of non-protein cell membrane such as glucide, lipids, and the like.

Description

technical field [0001] The invention relates to a method for screening disease markers. Background technique [0002] The occurrence and development of diseases lead to changes in the types and quantities of related molecules in cells, and these molecules closely related to specific diseases are disease markers. Discovering and searching for disease markers, and detecting and tracking these changes at the molecular level is a very effective way to understand the mechanism of disease, improve diagnostic accuracy, early diagnosis window period, formulate reasonable treatment plan and monitor the effect of treatment. The current method for finding disease markers is generally a proteomics method combining mass spectrometry and two-dimensional electrophoresis. However, this method has great limitations in the identification of membrane proteins, mainly because of the poor solubility of membrane proteins. About 30% of all proteins are membrane proteins, and currently less than ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/10C40B30/04C12P19/34C12Q1/48
Inventor 谭蔚泓上官棣华方晓红王柯敏杨朝勇
Owner 谭蔚泓
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