Reagent and method for separating and determining dissociative DNA in blood

A technology of reagents and kits, which is applied in the field of medical oncology, can solve the problems of unsuitability for quantitative analysis of free blood DNA and large errors, and achieve the effects of low cost, high DNA yield, and short DNA separation time

Inactive Publication Date: 2005-10-26
上海奇诺肿瘤生物高新技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] UV spectrophotometry (measurement of light absorption at 260nm wavelength) is commonly used for DNA quantification, but this method has too much error when the DNA conce

Method used

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  • Reagent and method for separating and determining dissociative DNA in blood
  • Reagent and method for separating and determining dissociative DNA in blood
  • Reagent and method for separating and determining dissociative DNA in blood

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: the establishment of real-time quantitative PCR detection method

[0037] 1.1 Materials and methods

[0038] A. Materials: Human A431 epithelioid cancer cell line was purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Fetal bovine serum and F12 medium were purchased from GIBCO. Tri Reagent RNA extraction kit was purchased from Molecular Research Center, USA. Revert Aid TM The first-strand cDNA synthesis kit was purchased from MBI Fermantas, Lithuania. PCR primers and fluorescent probes were synthesized by Shanghai Saibaisheng Gene Technology Co., Ltd. The sequencing of PCR products was completed by Treasure Bioengineering (Dalian) Company. Ex Taq DNA polymerase and pMD 18-T PCR product cloning vector and real-time quantitative PCR kit were purchased from Bao Biological Engineering (Dalian) Co., Ltd.

[0039] B. Design of primers and probes: According to the Genbank database, the human epithelial growth fa...

Embodiment 2

[0049] Example 2: Linear relationship of genomic DNA detection

[0050] 2.1 Materials and methods

[0051] 2 cases of normal lung tissue were surgical resection specimens of lung cancer. The lung tissue above 5cm away from the cancer tissue was selected and stored in liquid nitrogen. DNAezsol Genomic DNA Extraction Kit was purchased from Shanghai Shenergy Bocai Biotechnology Company.

[0052] Take 50 mg of lung tissue and extract DNA according to the operating instructions. The obtained genomic DNA is measured by ultraviolet spectrophotometry for A260 and A280, requiring A260 / A280>1.80. Then sample DNA was taken for serial dilution, the concentrations were 50, 25, 5, 2.5, 0.5 and 0.25 ng / μl, and 2 μl of each was taken for quantitative PCR detection.

[0053] 2.2 Results

[0054] image 3 The results showed that the real-time quantitative detection of genomic DNA in normal lung tissue had a detection limit of at least 0.5 ng / reaction, and there was a linear relationship betw...

Embodiment 3

[0055] Embodiment 3: Quantitative analysis of serum free DNA

[0056] 3.1 Materials and methods

[0057] There were 10 serum samples from healthy people and 42 serum samples from lung cancer patients. All patients were confirmed by cytology or histology. The serum preparation method is as follows: 3ml of blood is extracted intravenously, placed in a clean centrifuge tube overnight at 4°C, then centrifuged at 1500 rpm for 5 minutes, and the upper layer of serum is kept in a -70°C refrigerator.

[0058] The extraction of serum free DNA adopts the blood free DNA extraction reagent a) that above-mentioned embodiment 3 makes, and its operating steps are as follows:

[0059] 1. Add 500 microliters of blood cell-free DNA extraction reagent a) into 1.5ml Eppendorf tube, and preheat at 95°C for 5 minutes;

[0060] 2. Add 100-500 microliters of plasma, heat at 95°C for 5 minutes, and shake vigorously for 20 seconds;

[0061] 3. Centrifuge at 13,000rpm for 5min;

[0062] 4. Transfer ...

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Abstract

This invention provides a method and a solution of separation and extraction of dissociative DNA in blood; it also provides a reagent box which is used to quantificational detecting of dissociative DNA in blood. This invention can detect the dissociative DNA in blood fast and sensitively and obtain the result.

Description

technical field [0001] This application relates to the field of medical oncology. Specifically, the application relates to a reagent for separating and extracting DNA in blood (serum or plasma), and a method for separating, extracting and quantitatively detecting DNA in blood (serum or plasma). Background technique [0002] In the 1970s, European scholars reported for the first time that there was free (circulating) DNA in the blood (serum or plasma) of malignant tumor patients, and its content was about 10 times that of healthy people. This type of free DNA is a double-stranded DNA molecule ranging from 200bp to 21kb, most of which exist in the form of nucleosomes. Molecular biological studies have confirmed that the cell-free DNA in the blood has the same genetic variation as the genomic DNA of the primary tumor, such as oncogene mutation, microsatellite imbalance, and tumor suppressor gene promoter methylation, etc. It comes from tumors, and its sources generally includ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 董强刚
Owner 上海奇诺肿瘤生物高新技术有限公司
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