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Method for determining double minute chromosome sequences in human ovarian carcinoma cell line UACC-1598

A technology of UACC-1598 and ovarian cancer cells, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, etc., can solve the problems of not being able to provide microscopic information of double micro bodies, and achieve comprehensive understanding, high repeatability, and guaranteed The effect of completeness

Active Publication Date: 2013-10-09
HARBIN MEDICAL UNIVERSITY
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Problems solved by technology

[0004] The purpose of the present invention is to solve the existing research on the structure of the double microbody mainly by means of cytogenetics, and to detect the structure of the double microbody by using optical microscope, electron microscope, atomic force microscope and fluorescence in situ hybridization.
These methods all describe the double minute from the macroscopic level, that is, the chromosome level, and cannot provide the microscopic information of the double minute, and then provide a method for the sequence determination of the double minute in the human ovarian cancer cell line UACC-1598

Method used

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  • Method for determining double minute chromosome sequences in human ovarian carcinoma cell line UACC-1598
  • Method for determining double minute chromosome sequences in human ovarian carcinoma cell line UACC-1598
  • Method for determining double minute chromosome sequences in human ovarian carcinoma cell line UACC-1598

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Embodiment Construction

[0029] see figure 1 ~ Fig. 6, the technical solution of a double minute sequence determination method in the human ovarian cancer cell line UACC-1598 in this embodiment is realized in this way:

[0030] 1. Culture of human ovarian cancer cell UACC-1598 and separation of intact double microsomes by pulsed field gel electrophoresis

[0031] 1. Sample preparation

[0032] Human ovarian cancer cells UACC-1598 were cultured in RPMI-1640 medium containing 10% FBS in 5% CO 2 and cultured at 37°C. Collect human ovarian cancer cells UACC-1598 in the logarithmic growth phase with a confluence rate of 70% to 80%, count and resuspend in an appropriate amount of PBS to make the cell density about 1 to 2×10 8 cells / mL and incubate in a 37°C water bath. Add an equal volume of 1.4% low-melting point agarose at 37°C, mix well, pour into a clean mold, and place at 4°C for 10-15 minutes to solidify. Take the rubber block out of the mold and place it in 3-5 times the volume of cell lysate (0...

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Abstract

The invention provides a method for determining double minute chromosome sequences in the human ovarian carcinoma cell line UACC-1598. The method includes the following steps that: (1) human ovarian carcinoma cells UACC-1598 are cultured, and the pulsed-field gel electrophoretic separation of whole double minute chromosomes in the cells is carried out; and (2) the 454 technique of the Roche company is applied to determine the sequences of the obtained double minute chromosomes in the human ovarian carcinoma cell line UACC-1598. The electrophoretically separated high-purity DMs (double minute chromosomes) greatly guarantee the integrity of an experimental subject, the 454 high-throughput sequencing technique of Roche is more suitable for analysis because of the existence of the heterogeneity of the double minute chromosome sequences, high repeatability and the total size of only tens of megabasses, consequently, the overall perspective and characteristics of the double minute chromosome sequences can be thoroughly known, and the authenticity of the obtained sequences is guaranteed to a great extent.

Description

technical field [0001] The invention relates to a method for determining double minute sequences in human ovarian cancer cell line UACC-1598, and belongs to the technical field of double minute sequence determination. Background technique [0002] Double minute chromosomes (DMs) are circular chromosomes that exist in pairs in tumor cells, do not contain centromeres, and can replicate autonomously. They are one of the main manifestations of gene amplification in tumor cells. Double microsomes exist in tumor cells and some cells treated with chemotherapy drugs, but no double microsomes are detected in normal cells. Since the double minute was first discovered in human colon cancer cells in 1962, it has been detected in most solid tumors and hematological tumors. The results of clinical specimen testing showed that double microsomes existed in 18% of breast cancer, 29% of ovarian cancer, 44% of colon cancer, 20%-30% of small cell lung cancer and 12.5% ​​of hematological tumors...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 傅松滨于旸朱静孟祥宁孙文靖张春玉
Owner HARBIN MEDICAL UNIVERSITY
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