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Apparent modification detection primer pair based on ATP4B genosome DNA and kit thereof

A gene body and kit technology, applied in the field of gene body DNA methylation modification detection, can solve problems such as inability to up-regulate

Inactive Publication Date: 2017-05-17
BEIJING JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have found that although DATS can up-regulate the expression of MT2A in gastric cancer, it cannot up-regulate the expression of ATP4B ( Figure 4 E) (Pan, Y., et al., Antioxid Redox Signal, 2016.)

Method used

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  • Apparent modification detection primer pair based on ATP4B genosome DNA and kit thereof
  • Apparent modification detection primer pair based on ATP4B genosome DNA and kit thereof
  • Apparent modification detection primer pair based on ATP4B genosome DNA and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Design of special primers for ATP4B gene DNA methylation

[0040] According to the distribution characteristics of DNA methylation modification in the genome (Maunakea AK., et al. Nature 2010, 466(7303); Jjingo D., et al. Oncotarget 2012; 3(4).), through manual comparison analysis, Select the DNA region rich in CG in the specific ATP4B gene sequence from GenBank, design multiple primer pairs with the software Primer Express 3.0, and screen out the following methylated primers and non-methylated primers. All primers are provided by Tianyi Huiyuan Technology Company synthesis.

[0041] The nucleotide sequence of the upstream primer of the methylated primer is: 5'-GAGTTTTAGCGTTATTGTTGGAATTCGGATAC-3' (shown in SEQ ID No. 3), and the nucleotide sequence of the downstream primer is: 5'-GTACCCCACCGAAACAAAATACG-3' (such as SEQ ID No. 4); the nucleotide sequence of the upstream primer of the unmethylated primer is: 5'-GGAGTTTTAGTGTTATTGTTGGAATTTGGATATG-3' (as shown in SEQ...

Embodiment 2

[0042] Example 2: Composition of a kit for detecting ATP4B gene DNA methylation modification

[0043] A kit for detecting ATP4B gene DNA methylation modification, that is, a kit for early diagnosis of gastric cancer, detection of chemotherapy sensitivity and detection of drug targets, including nucleotide sequences such as SEQ ID No. 3 and SEQ ID The methylated primer shown in No.4, the nucleotide sequence of the unmethylated primer shown in SEQ ID No.5 and SEQ ID No.6, the methylated control DNA template for MSP (modified by sulfurization) Afterwards, more than 90% hypermethylated DNA appears in the ATP4B gene body region), unmethylated control DNA template (ATP4B gene body region shows unmethylated normal tissue cell DNA), deionized water as a negative system control, PCR Reaction reagents.

Embodiment 3

[0044] Example 3: Detection of ATP4B methylation in cells and tissues

[0045] (1) The extraction process of cell line DNA

[0046] The gastric cancer cell lines and normal gastric cell lines in good growth condition and in the logarithmic growth phase were digested with trypsin and then counted, and collected in a 1.5mL centrifuge tube. The number of cells was about 10 5 To 10 6 One. Centrifuge at 13000 rpm for 10 seconds, discard the supernatant and add 200 μL 1×PBS to resuspend the cells. After washing again, resuspend the cells in 180 μL 1×PBS. The following steps refer to the Biomed Genomic DNA Rapid Extraction Kit Operation Manual. The specific operations are as follows:

[0047] Add 20 μL of 20 mg / mL proteinase K to the cell suspension, mix well, add 200 μL of binding solution, and place at 70°C for 10 minutes. After cooling, add 100 μL of isopropanol, add the mixture to the adsorption column, centrifuge at 13000 rpm for 30 seconds, and discard the waste liquid. Add 500μL of...

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Abstract

The invention discloses an apparent modification detection primer pair based on ATP4B genosome DNA and a kit thereof. The invention provides an application of ATP4B genosome DNA fragment as a molecular marker or a medicine effect target shown as a nucleotide sequence of SEQ ID No.1 or SEQ ID No.2 and in preparation of relative detection kits; and further provides a molecular marker and a kit used for stomach cancer early diagnosis and chemotherapy sensitive detection. The kit comprises a methylation primer shown as a SEQ ID No.3 and SEQ ID No.4 and a non-methylation primer shown as SEQ ID No.5 and SEQ ID No.6. The obtained detection result can monitor stomach cancer patient condition and individualized medication, perform early warning for tumor, and provide an evaluation method for prognosis after medical treatment.

Description

Technical field [0001] The invention relates to a method for detecting DNA methylation modification of gene body. More specifically, it relates to a primer pair and a kit for detecting ATP4B gene body DNA methylation modification based on PCR. Background technique [0002] Gastric cancer is one of the most important malignant tumors in the world. It is still the third most deadly cancer in the world, and its incidence in Asia is increasing (Bray F, et al. Lancet Oncol 13:790-801, 2012; Ushijima, T., J Biochem Mol Biol, 2007.40(2): p.142-50.), due to the lack of effective detection methods for early detection, more than 90% of patients are in the middle and late stages. For patients in the middle and advanced stages, the clinical treatment is mostly chemotherapy. Although such treatment can inhibit the development of tumors to a certain extent, the side effects of chemotherapy and the tolerance of patients to chemotherapy drugs in later stages cannot be ignored. Problems that ne...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/106C12Q2600/154
Inventor 黄家强林舒晔林博楠王晓月
Owner BEIJING JIAOTONG UNIV
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