Method of purifying paraffin DNA and application of paraffin DNA to genomics

A purification method and paraffin embedding technology, applied in the field of genetic engineering, can solve the problems of poor quality, unimproved DNA yield and quality, low yield of paraffin DNA, etc., to achieve the effect of reducing content, reducing damage and binding, and promoting protein degradation

Active Publication Date: 2016-01-27
BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In our early exploration stage, we found that the paraffin DNA extracted by these schemes has the general characteristics of low yield and poor quality (Fassunke, J., et al., Utility of different massive parallel sequencing platforms formation profiling clinical samples and identification of pitfalls using FFPE tissue. IntJMolMed, 2015)
At present, there is no specific solution for the whole process of paraffin section production to improve the yield and quality of DNA extraction

Method used

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  • Method of purifying paraffin DNA and application of paraffin DNA to genomics
  • Method of purifying paraffin DNA and application of paraffin DNA to genomics
  • Method of purifying paraffin DNA and application of paraffin DNA to genomics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] 1. Place the kidney cancer paraffin sample (paraffin shavings) at room temperature (20-25°) for 2 hours.

[0028] 2. Weigh ≤25mg of paraffin shavings tissue and put it into a 2ml EP tube.

[0029] 3. Add 1200ul xylene; shake and mix for 10 seconds; place on a rotator for 10 minutes at room temperature.

[0030] 4. Centrifuge at 10000G for 70 seconds at room temperature.

[0031] 5. Discard the supernatant.

[0032] 6. Repeat steps 3-5 once.

[0033] 7. Add 1200ul 100% ethanol, shake and mix for 15 seconds.

[0034] 8. Centrifuge at 11000G for 70 seconds at room temperature.

[0035] 9. Discard the supernatant (do not remove any pellet).

[0036] 10. Repeat steps 7-9 once.

[0037] 11.37 ° water bath 2mlEP tube for 30 minutes.

[0038] 12. Add 100ul of 0.1M, 0.2M, 0.5M or 1.0M sodium borohydride solution (set as Group 2, 3, 4, 5 respectively).

[0039] 13. 50 ° water bath 2mlEP tube for 30 minutes. (The control group does not add sodium borohydride, without step...

Embodiment 2

[0058] 1. Place the kidney cancer paraffin sample (paraffin shavings) at room temperature (20-25°) for 2 hours.

[0059] 2. Weigh ≤25mg of paraffin shavings tissue and put it into a 2ml EP tube.

[0060] 3. Add 1200ul xylene; shake and mix for 15 seconds; place on a rotator for 10 minutes at room temperature.

[0061] 4. Centrifuge at 10000G for 70 seconds at room temperature.

[0062] 5. Discard the supernatant.

[0063] 6. Repeat steps 3-5 twice.

[0064] 7. Add 1200ul 100% ethanol, shake and mix for 15 seconds.

[0065] 8. Centrifuge at 11000G for 70 seconds at room temperature.

[0066] 9. Discard the supernatant (do not remove any pellet).

[0067] 10. Repeat steps 7-9 once.

[0068] 11.37 ° water bath 2mlEP tube for 30 minutes.

[0069] 12. Add 100ul 0.2M sodium borohydride solution.

[0070] 13. 50 ° water bath 2mlEP tube for 30 minutes.

[0071] 14. Add 180ul ATL, 40ul proteinase K; shake and mix for 20 seconds.

[0072] 15.61.5 ° water bath for 48-72 hours, ...

Embodiment 3

[0104] 1. Liver cancer, lymph node, prostate cancer, and benign prostatic hyperplasia samples (paraffin shavings) (respectively set as experimental groups 1-4) were placed at room temperature (20-25°) for 2 hours.

[0105] 2. Weigh ≤25mg of paraffin shavings tissue and put it into a 2ml EP tube.

[0106] 3. Add 1200ul xylene; shake and mix for 15 seconds; place on a rotator for 10 minutes at room temperature.

[0107] 4. Centrifuge at 10000G for 70 seconds at room temperature.

[0108] 5. Discard the supernatant.

[0109] 6. Repeat steps 3-5 once.

[0110] 7. Add 1200ul 100% ethanol, shake and mix for 15 seconds.

[0111] 8. Centrifuge at 11000G for 70 seconds at room temperature.

[0112] 9. Discard the supernatant (do not remove any pellet).

[0113] 10. Repeat steps 7-9 once.

[0114] 11.37 ° water bath 2mlEP tube for 30 minutes.

[0115] 12. Add 100ul 0.2M sodium borohydride solution.

[0116] 13. 50 ° water bath 2mlEP tube for 30 minutes.

[0117] 14. Add 180ul A...

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PUM

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Abstract

The invention provides a method of purifying DNA in a paraffin embedding sample. The method comprises the steps that (1) the sample is dewaxed; (2) formaldehyde in the sample is removed; (3) cell lysis is conducted; (4) the DNA is extracted; (5) the DNA and protein are disconnected; (6) the DNA is purified; in the step (1), xylene is used for dewaxing the sample. The purity of the DNA obtained through the method is high, and the sequencing quality is approximate to that of a DNA sample of fresh tissue.

Description

technical field [0001] The invention relates to a paraffin DNA purification method and the application of the method in genomics, in particular to a method for purifying DNA from paraffin-embedded human tissue samples, which belongs to the field of genetic engineering. Background technique [0002] The rapid development of genomics brings opportunities for precision medicine. Taking the individualized treatment of tumors as an example, the paraffin specimens of tumors preserved in the pathology department of the hospital are the prerequisite for finding drug targets and realizing precision medicine for cancer patients. However, the processing procedures (such as formalin immersion, paraffin embedding, etc.) experienced by paraffin specimens during the production process are the main reasons for the high degree of degradation of paraffin DNA. The degraded paraffin DNA can only be used to a limited extent in the study of tumor genomics—the DNA quality is poor (260 / 280 ratio i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68
Inventor 陈科慈维敏
Owner BEIJING INST OF GENOMICS CHINESE ACAD OF SCI CHINA NAT CENT FOR BIOINFORMATION
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