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Method for separation and purification of malignant tumor cell dna by cationic montmorillonite

A cationic, malignant tumor technology, applied in the field of separation and purification of malignant tumor cell DNA, can solve the problems of inconvenient high-throughput operation, single purification model, and high cost of material synthesis, so as to reduce the preparation cost, improve the dispersion effect, and enhance the adsorption. effect of action

Active Publication Date: 2021-12-24
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problems of single purification model, inconvenient high-throughput operation and high cost of material synthesis in the existing diagnostic kits, the present invention proposes the application of a cationic montmorillonite in the separation and purification of malignant tumor cell DNA. The low-cost montmorillonite is modified, and the cationic montmorillonite obtained after modification can quickly and efficiently purify the DNA of malignant tumor cells. Structure of DNA in malignant tumor cells wreaks havoc

Method used

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  • Method for separation and purification of malignant tumor cell dna by cationic montmorillonite
  • Method for separation and purification of malignant tumor cell dna by cationic montmorillonite
  • Method for separation and purification of malignant tumor cell dna by cationic montmorillonite

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Cell Lysis:

[0028] Culture HeLa cells in a 6cm-diameter petri dish, then add 500 μL of 2% sodium dodecyl sulfate solution to lyse, transfer the cell lysate into an EP tube and heat at 80°C for 5 min, at an amplitude of 20%, Sonicate at a power of 500W and store in a -20°C refrigerator for later use.

[0029] Preparation of lithium-ion montmorillonite:

[0030] Dissolve Li in 9 mL of deionized water 2 CO 3 0.23 g, add 1.00 g of calcium-based montmorillonite, react in a constant temperature water bath at 60 °C for 24 h, centrifuge, dry at 60 °C for 24 h, and store in a dry dish in an airtight manner, marked as 0.5Li-MMT.

[0031] DNA adsorption:

[0032] Take 70 μL of 30 g / L lithium-ion montmorillonite suspension, add 20 μL of cell lysate and 10 μL of sodium acetate solution with a pH of 5.0, react for 2 minutes and centrifuge at a centripetal acceleration of 5000 g for 2 minutes, take the supernatant and wash with agar The DNA content was determined by gel electro...

Embodiment 2

[0036] Cell Lysis:

[0037] Culture HeLa cells in a petri dish with a diameter of 6 cm, then add 500 μL of 2% sodium dodecyl sulfate solution to lyse, transfer the cell lysate into an EP tube and heat at 90°C for 5 min, at an amplitude of 20%, Sonicate at a power of 500W and store in a -20°C refrigerator for later use.

[0038] Preparation of lithium-ion montmorillonite:

[0039] Dissolve Li in 9 mL of deionized water 2 CO 3 0.47 g, add 1.00 g of calcium-based montmorillonite, react in a constant temperature water bath at 60 °C for 24 h, centrifuge, dry at 60 °C for 24 h, and store in a dry dish in an airtight manner, marked as Li-MMT.

[0040] DNA adsorption:

[0041] Take 70 μL of 30 g / L lithium-ion montmorillonite suspension, add 20 μL of cell lysate and 10 μL of sodium acetate solution with a pH of 5.0, react for 2 minutes and centrifuge at a centripetal acceleration of 5000 g for 2 minutes, take the supernatant and wash with agar The DNA content was determined by gel...

Embodiment 3

[0045] Cell Lysis:

[0046] Culture HeLa cells in a 6cm-diameter petri dish, then add 500 μL of 2% sodium dodecyl sulfate solution for lysis, transfer the cell lysate into an EP tube and heat at 100°C for 5 minutes, at an amplitude of 20%, Sonicate at a power of 500W and store in a -20°C refrigerator for later use.

[0047] Preparation of lithium-ion montmorillonite:

[0048] Dissolve Li in 9 mL of deionized water 2 CO 30.71 g, add 1.00 g of calcium-based montmorillonite, react in a constant temperature water bath at 60 °C for 24 h, centrifuge, dry at 60 °C for 24 h, and store in a dry dish in an airtight manner, marked as 1.5Li-MMT.

[0049] DNA adsorption:

[0050] Take 70 μL of 30 g / L lithium-ion montmorillonite suspension, add 20 μL of cell lysate and 10 μL of sodium acetate solution with a pH of 5.0, react for 2 minutes, and then centrifuge at a centripetal acceleration of 5000 g for 2 minutes. The supernatant was taken and the DNA content was determined by agarose g...

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Abstract

The invention relates to the application of a cationic montmorillonite in the separation and purification of malignant tumor cell DNA. HeLa cells are cracked by using sodium dodecyl sulfate solution, and the cell lysate is obtained after heating and ultrasonication, and then the cationic Type montmorillonite suspension, adjust the pH to 5.0-6.0 with sodium acetate, add it to the cell lysate for DNA adsorption, centrifuge after adsorption, wash the lower precipitate with the eluent for DNA desorption. In the present invention, by modifying the low-cost montmorillonite, the modified cationic montmorillonite can quickly and efficiently purify the DNA of malignant tumor cells. The process does not cause damage to the structure of the DNA of the malignant tumor cells.

Description

technical field [0001] The invention relates to the separation and purification of malignant tumor cell DNA, in particular to the application of a cationic montmorillonite in the separation and purification of malignant tumor cell DNA. Background technique [0002] Tumor is one of the diseases that seriously endanger human health. After necrosis and apoptosis, tumor cells release tumor genome free DNA fragments, called ctDNA, which can continuously circulate in the human blood system. ctDNA can be used non-invasively for the early diagnosis, development trend and prognosis judgment of tumors, and is a tumor marker with broad application prospects. One 100g (3×10 10 cancer cells), about 3.3% of tumor cell DNA enters the blood circulation every day. As the carrier of genetic information, DNA stores important information related to human diseases. Realizing the detection of malignant tumor cell DNA can predict the human body's disease trend and potential hidden dangers. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/10
CPCC12N15/1006C12Q2523/308
Inventor 张毅杨华明谢虹忆
Owner CENT SOUTH UNIV
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