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Whole-genom sifting method for BPDE carcinogen related gene

A genome-wide, screening method technology used in the field of bioengineering

Inactive Publication Date: 2008-01-30
INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The combination of AFLP technology and IMS technology in the screening of tumor susceptibility genes has not been reported yet.

Method used

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  • Whole-genom sifting method for BPDE carcinogen related gene
  • Whole-genom sifting method for BPDE carcinogen related gene
  • Whole-genom sifting method for BPDE carcinogen related gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0112] A genome-wide screening method for BPDE carcinogenic genes, including the following steps:

[0113] (1) Extract genomic DNA from normal lung tissue

[0114] (2) Preparation of AFLP DNA fragments: AFLP DNA fragments are obtained by double digestion of genomic DNA;

[0115] (3) Immunomagnetic enrichment and separation of AFLP DNA fragments interacting with BPDE: AFLP DNA fragments are incubated with BPDE to form AFLP DNA-BPDE adducts, and immune nanomagnetic particles directed against BPDE antigens are added. Enrichment and separation of AFLP DNA fragments that interact with BPDE;

[0116] (4) AFLP PCR amplification: the connection of AFLP DNA fragments and adaptors, pre-amplification and selective amplification;

[0117] (5) Denaturing polyacrylamide gel electrophoresis and silver staining of PCR products selectively amplified by AFLP;

[0118] (6) Differential fragment recovery, amplification, cloning, sequencing and homology similarity search analysis.

Embodiment 2

[0120] Extraction of genomic DNA from mouse lung tissue

[0121] 1. Main instruments

[0122] Desktop high-speed refrigerated centrifuge (UNIVERSAL 32R, Hettich, Germany), Beckman Du530 spectrophotometer (Beckman, Germany), UV analyzer (Shanghai Kanghua Instrument Manufacturing Plant), high-purity water device SCD-II (Institute of Medical Equipment, Academy of Military Medical Sciences) )

[0123] 2. Main materials and reagents

[0124] BALB / c mice (Experimental Animal Center of the Academy of Military Medical Sciences), DNA extraction kit (Beijing Tianwei Times), Proteinase K (Dalian Bao Biological Engineering Co., Ltd.), absolute ethanol (AR, Tianjin Shentai Chemical Reagent Co., Ltd.) ), Agarose (Dalian Bao Biological Engineering Co., Ltd.), DNA Marker (Dalian Bao Biological Engineering Co., Ltd.)

[0125] 3. Preparation of main reagents

[0126] (1) Preparation of PBS pH7.4: NaCl8.0g, KCl 0.2g, KH 2 PO 4 0.2g, Na 2 HPO 4 ·12H2 O 2.9g, 900ml ultrapure water, adjust the pH value...

Embodiment 3

[0145] Double enzyme digestion to prepare AFLP DNA fragments

[0146] 1. Main instruments

[0147] Electric heating constant temperature water bath (Tianjin North China Experimental Instrument Factory)

[0148] 2. Main reagents

[0149] Mouse lung tissue DNA, Taq I endonuclease (Invitrogen, USA), Pst I endonuclease (Invitrogen, USA)

[0150] 3. Method steps

[0151] (1) First digest with Taq I endonuclease. The tube without endonuclease was used as a control.

[0152] The reaction system is as follows:

[0153] Taq I 1μl

[0154] 10×buffer 2μl

[0155] 0.1%BSA 2μl

[0156] DNA 1μl

[0157] ddH 2 O 14μl

[0158] Total 20μl

[0159] Reaction conditions: put in a constant temperature water bath, digest in a constant temperature water bath at 65°C for 2h;

[0160] (2) Then perform the following operations to add 5μl of the reaction solution containing Pst I to each reaction system:

[0161] Pst I 1μl

[0162] 10×buffer 2μl

[0163] ddH 2 O 2μl

[0164] Total 5μl

[0165] The total vol...

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Abstract

The invention discloses a whole genome screening method for screening relative BPDE oncogenes and comprises the steps that: (1) genome DNA is extracted from normal lung tissue; (2) AFLP DNA fragment is prepared; (3) immunomagnetic of AFLP DNA fragment interacting with BPDE is enriched and separated; (4) AFLP PCR is increased; (5) denatured polyacrylamide gel electrophoresis and silver stain visualization are done to selective PCR amplification products of AFLP; (6) differential fragment is recycled, amplified, cloned and tested for sequence and homologous similarity and analysis are done. The whole genome screening method for screening relative BPDE oncogenes of the invention is the high effective and specific method and more BPDE susceptible oncogenes are screened in the whole genome level, which provides a basis for stating BPDE carcinogenic molecular mechanism.

Description

Technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a whole genome screening method of BPDE carcinogenic related genes. Background technique [0002] With the rapid development of social economy, environmental pollution has become a serious problem faced by people. People living in industrial countries or regions are generally facing the risk of mutagenesis, carcinogenesis or teratogenicity caused by chemical substances. Tumors, especially malignant tumors, seriously endanger human health and life. Many countries have invested huge manpower and material resources for this, but the harm of tumors has not been curbed so far. In recent years, the incidence of malignant tumors has been rising in many countries. In my country, the mortality rate of malignant tumors has ranked first in the cause of death. Lung cancer is the most common malignant tumor of the respiratory system. The morbidity and mortality of lung cancer in my country...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 陈照立李君文王景峰金敏王新为谌志强邱志刚
Owner INST OF HYGIENE & ENVIRONMENTAL MEDICINE PLA ACAD OF MILITARY MEDICAL
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