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Method for extracting DNA from old formalin-fixed tissues

A formalin and tissue technology, applied in the field of molecular biology detection, can solve problems such as lack of method stability and achieve high purity

Inactive Publication Date: 2010-07-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the critical point drying method can be used for DNA extraction of old fixed tissues, it is mainly limited to the extraction of animal and plant specimens, and requires special instruments, and the stability of the method is also lacking.

Method used

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  • Method for extracting DNA from old formalin-fixed tissues
  • Method for extracting DNA from old formalin-fixed tissues
  • Method for extracting DNA from old formalin-fixed tissues

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1) Take 45mg of tissue, put it into a clean 5ml test tube, add 2.5ml of ddH2O into the test tube, put the test tube into a beaker, add water into the beaker, the amount of water should be just above the liquid level of the test tube, microwave Heating with 500W power for 3 minutes, replacing the water in the test tube, then heating with 500W power microwave for 2 minutes, and taking out the tissue;

[0025] 2) Cut the tissue into pieces, blot dry with filter paper, and soak in ethanol with gradient concentrations in sequence: 70% for 2 hours; 80% for 0.5 hours; 90% for 0.5 hours; 95% for 0.5 hours; 100% for 0.5 hours;

[0026] 3) Soak in 0.5×phosphate buffer solution twice for 0.5 hour, filter paper to absorb the phosphate buffer solution;

[0027] 4) Add 600 μl of ordinary tissue lysate, grind it into a homogenate, transfer it into a 2ml centrifuge tube, add 15 μl of proteinase K at a concentration of 20 mg / ml, and digest in a water bath at 55°C until the solution beco...

Embodiment 2

[0035] 1) Take 55mg of tissue, put it into a clean 5ml test tube, add 3ml of ddH2O into the test tube, put the test tube into a beaker, add water into the beaker, the amount of water should be just above the liquid level of the test tube, microwave 500W Power heating for 3 minutes, replace the water in the test tube, then microwave heating with 500W power for 2 minutes, and take out the tissue;

[0036] 2) Cut the tissue into pieces, blot dry with filter paper, and soak in ethanol with gradient concentrations in sequence: 70% for 2 hours; 80% for 0.5 hours; 90% for 0.5 hours; 95% for 0.5 hours; 100% for 0.5 hours;

[0037] 3) Soak in 0.5×phosphate buffer solution twice for 0.5 hours, and absorb the phosphate buffer solution through filter paper;

[0038] 4) Add 1000 μl of ordinary tissue lysate, grind it into a homogenate, transfer it into a 2ml centrifuge tube, add 20 μl of proteinase K at a concentration of 20 mg / ml, and digest in a water bath at 55°C until the solution beco...

Embodiment 3

[0046] 1) Take 50mg of tissue, put it into a clean 5ml test tube, add 2.75ml of ddH2O into the test tube, put the test tube into a beaker, add water into the beaker, the amount of water should be just above the liquid level of the test tube, microwave Heating with 500W power for 3 minutes, replacing the water in the test tube, then heating with 500W power microwave for 2 minutes, and taking out the tissue;

[0047] 2) Cut the tissue into pieces, blot dry with filter paper, and soak in ethanol with gradient concentrations in sequence: 70% for 2 hours; 80% for 0.5 hours; 90% for 0.5 hours; 95% for 0.5 hours; 100% for 0.5 hours;

[0048] 3) Soak in 0.5×phosphate buffer solution twice for 0.5 hours, and absorb the phosphate buffer solution through filter paper;

[0049] 4) Add 800 μl of ordinary tissue lysate, grind it into a homogenate, transfer it into a 2ml centrifuge tube, add 17.5 μl of proteinase K at a concentration of 20 mg / ml, and digest in a water bath at 55°C until the ...

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Abstract

The invention discloses a method for extracting DNA from old formalin-fixed tissues. The basic principle of the method is to utilize the microwave thermal effect for fast removing formalin in the fixed tissues via water molecules, and eliminate the DNA-protein crosslinking caused by aldehyde groups of the formalin, thereby completely or partially recovering a DNA structure and further obtaining the DNA with better quality. The invention relates to the simple and high-efficient method for extracting the DNA from the old formalin-fixed tissues, which is based on microwave thermal remediation and can obtain the DNA with higher quality (large fragments and high purity), so that the method can be used for multiplex fluorescent PCR-capillary electrophoresis (FM-CE), denaturing high performance liquid chromatography (DHPLC) analysis, direct DNA sequencing and other molecular biological method analyses.

Description

technical field [0001] The invention relates to a method for extracting DNA from old formalin-fixed tissues, which is used for molecular biology detection. Background technique [0002] With the completion of the Human Genome and Haploid Project, life science research has entered a new stage of functional genomics. Emerging disciplines represented by cancer genomics and pharmacogenomics have brought bright prospects for the realization of individualized prevention and treatment of major human diseases such as cancer. However, the first prerequisite for these large-scale genomics tests is the availability of good-quality DNA samples. Fresh tissue specimens are an ideal source of DNA samples, but sampling and transportation are difficult, and may affect the final pathological diagnosis of patients and affect the quality of medical care, so it is difficult to carry out routinely. Paraffin-embedded tissue (wax block) is routinely preserved in the pathology department of the ho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C07H21/04C07H1/06
Inventor 吕炳建钱昕盛弘强来茂德
Owner ZHEJIANG UNIV
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