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Kit for detection of gene expression of cytosine deaminase and related molecules in free dna of peripheral blood and its primer pair

A cytosine deaminase and gene expression technology, applied in the field of detection kits and primer pairs for detection of cytosine deaminase and its related molecular gene expression in free DNA in peripheral blood, can solve the heterogeneity of tumor cells , mutation, low activity and other problems, to achieve the effect of improving sensitivity

Active Publication Date: 2017-11-21
欧浦德生物科技(北京)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] In addition, UNG, as an important enzyme molecule for cells to repair cytosine mutations, is often highly expressed in tumor cells, but its activity is not high, and it cannot effectively repair mutations caused by cytosine deamination
[0008] It can be seen that in the process of cell evolution, the activity of the invader gene is mutated to limit its activity, but at the same time, as a double-edged sword, it also increases the risk of its own DNA mutation, and its continuous high expression is the cause of cell transformation and tumors. An important cause of progression and a source of tumor cell heterogeneity

Method used

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  • Kit for detection of gene expression of cytosine deaminase and related molecules in free dna of peripheral blood and its primer pair
  • Kit for detection of gene expression of cytosine deaminase and related molecules in free dna of peripheral blood and its primer pair
  • Kit for detection of gene expression of cytosine deaminase and related molecules in free dna of peripheral blood and its primer pair

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Embodiment 1: Establish detection method

[0065] 1) Sample preparation:

[0066] Extract peripheral blood and extract cell-free DNA;

[0067] Use HpaII endonuclease to digest free peripheral blood DNA. The enzyme digestion system is shown in Table 1; the enzyme digestion condition is: 37°C for 2 hours; after HpaII enzyme digestion, further inactivate the enzyme digestion reaction system , the inactivation condition is: inactivation at 65°C for 20min. The inactivated products are used as templates for real-time fluorescent quantitative PCR (qPCR) amplification reactions of Apobec3B / C, Apobec3F / G and UNG genes.

[0068] Use NheI endonuclease to digest free peripheral blood DNA. The enzyme digestion system is shown in Table 2; the enzyme digestion conditions are: 37°C for 2 hours; after NheI enzyme digestion, further inactivate the enzyme digestion reaction system, the inactivation conditions For: 65 ℃ inactivation 20min. The inactivated product is used as a template ...

Embodiment 2

[0124] According to the method of implementation 1, the Apobec3B / C, Apobec3F / G, UNG and AID gene mutations in normal people and tumor patients were detected, and the ΔΔCt weighted value was calculated. The △△Ct weighted value of cancer patients is shown in Table 12, and the △△Ct weighted value of cancer patients is shown in Table 13.

[0125] Table 12: Control group (30 cases):

[0126]

[0127]

[0128] Table 13: Patients in Tumor Group

[0129]

[0130]

[0131] There were 11 cases of lung cancer, 10 cases of liver cancer, 9 cases of gastric cancer, 5 cases of colorectal cancer, 3 cases of esophageal cancer, 2 cases of pancreatic cancer, 1 case of breast cancer, and 10 cases of other tumors.

[0132] According to statistical analysis, the Cut Off value was set to 13, so that 6 cases (samples 8, 12, 14, 17, 20 and 23) of 30 normal subjects had a slightly higher △△Ct weighted value than 13; There were 4 cases (samples 8, 9, 38 and 40) whose ΔΔCt weighted value wa...

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Abstract

The invention discloses a kit for detecting gene expression of cytosine deaminase (Apobec3B / C, Apobec3F / G, AID) and related molecules (UNG) in free DNA of peripheral blood and a primer pair set thereof. As shown in the sequence table SEQ ID No: 1 to SEQ ID No: 8. The kit containing this primer pair can evaluate the risk of cancer and the effect of treatment by detecting the methylation modification of Apobec3B / C, Apobec3F / G, AID and UNG gene promoters. This detection method has high sensitivity and specificity , directness, real-time and stability characteristics.

Description

technical field [0001] The invention relates to a kit for tumor screening, curative effect evaluation and prognosis prediction by using peripheral blood free DNA. It belongs to the field of biotechnology. Background technique [0002] With the increasing incidence of tumors year by year, tumors are becoming a normal disease, and the effectiveness of tumor treatment depends on early detection. At present, the screening methods of tumors mainly rely on imaging, but imaging is limited by physical constraints. Due to the limitation of resolution, it is difficult to detect tumors at an early stage, so there is an embarrassing situation where either the test results are normal or they are already in the middle and advanced stages. Molecular testing came into being. Traditional tumor markers are mainly proteins related to tumor metabolism. Clinical data over the years have shown that their sensitivity and specificity are low, and these indicators do not change in some tumor patie...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/34C12N15/11
CPCC12Q1/6886C12Q2600/118C12Q2600/154
Inventor 朱运峰刘芳田红池吕程程
Owner 欧浦德生物科技(北京)有限公司
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