Histone acetylation modified MT2A genome detection kit and primer pairs

A detection kit and primer pair technology, applied in the field of genome detection

Inactive Publication Date: 2017-03-29
BEIJING JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These studies suggest that MT2A may be used as a new epigenetic regulatory marker and target for the prediction and detection of chemotherapy-sensitive treatment options for gastric cancer, but there is no reliable and sensitive method for detecting the epigenetic status of MT2A genome DNA group method for predicting the effectiveness of treatment regimens

Method used

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  • Histone acetylation modified MT2A genome detection kit and primer pairs
  • Histone acetylation modified MT2A genome detection kit and primer pairs
  • Histone acetylation modified MT2A genome detection kit and primer pairs

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: Design of special primers for MT2A genomic DNA histone acetylation

[0086]According to the distribution characteristics of histone modification in the genome, the specific MT2A gene sequence was selected from GenBank, and the DNAMAN 6.0.40 software was used for comparison and analysis to find out the conserved gene sequence of MT2A, and the primers were designed with the software Primer Express 3.0, and the upstream primer MT2A-FP : the nucleotide sequence is: 5'-AGGTTATTCGGAGCCCCCTT-3', (SEQ ID No.1), the downstream primer MT2A-RP: the nucleotide sequence is: 5'-GTTCCTCAGTCCACAACCGA-3', (SEQ ID No.2) ;

[0087] All primers were synthesized by Tianyi Huiyuan Technology Co., Ltd.

Embodiment 2

[0088] Example 2: The composition of the detection kit for MT2A genomic DNA histone acetylation

[0089] MT2A genomic DNA histone acetylation detection kit, which is shown in the primer pair shown in the sequence table SEQ ID No.1 to SEQ ID No.2, as shown in the sequence table SEQ ID No.3 to SEQ ID No.4 Positive control primer pair, chromatin immunoprecipitation reagent set, nucleic acid extraction reagent and PCR reaction reagent.

[0090] The primers used to detect histone acetylation of MT2A genomic DNA are shown in Example 1;

[0091] 1% formalin, PBS, lysis buffer, dilution buffer, TSE I, TSE II, Buffer III, TE buffer, elution buffer of the chromatin immunoprecipitation reagent group, the preparation methods of the above reagents as follows:

[0092] Dilute 1% formalin: 37% formaldehyde with PBS at a volume ratio of 1:36.

[0093] Lysis buffer: 1% SDS, 5mM EDTA, 50mM Tris.HCl[pH 8.0], protease inhibitor cocktail;

[0094] Dilution buffer: 1% Triton X-100, 2mM EDTA, 15...

Embodiment 3

[0103] Example 3: Specificity test of MT2A genomic DNA histone acetylation

[0104] 1. Preparation of samples

[0105] Gastric cancer cells were inoculated in a 10cm-diameter culture dish, and when the cells grew to about 60-80%, DATS (40 μM) was added for 12 hours, normal saline was added to the negative control group, and TSA (5 nM) was added to the positive control group for 24 hours. Collect 3-5 Petri dishes per group.

[0106] 2. Collect and wash samples

[0107] The cells were washed 1-2 times with room temperature pre-warmed PBS, and then treated with 1% formalin at 37°C for 10 min. Wash the cells twice with ice-cold PBS, then transfer to 1ml of ice-cold PBS with a cell scraper, centrifuge at 3,000rpm at 4°C for 2min, and discard the supernatant. Resuspend the cell pellet with 400 μl lysis buffer and incubate on ice for 10 min.

[0108] 3. Ultrasonic fragmentation of chromatin DNA, sonication 16 times, 2s each time, centrifugation at 4°C, 14,000g for 15min, and coll...

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Abstract

The invention discloses a histone acetylation modified MT2A genome detection kit and primer pairs. The detection kit comprises primer pairs as shown in SEQ ID No:2 to SEQ ID No:2 of a sequence table, a positive primer pair, a chromatin Immunoprecipitation assay kit, a nucleic acid extracting reagent and a PCR reaction agent. The histone acetylation modified MT2A genome detection kit can be used for specficially detecting histone acetylation level associated with DNA of a MT2A genome; meanwhile, the reagent kit also can be used for carrying out peculiar and quantitative detection on the genome histone acetylation level associated with the DNA of the MT2A genome; and the detected results can provide basis for establishing a scheme for treating cancer patients, and also provides an evaluation method for prognosis, on drug therapy, of the cancer patients.

Description

technical field [0001] The present invention relates to a genome detection for histone acetylation modification, more specifically, relates to the gene detection of MT2A promoter region related to histone acetylation modification. Background technique [0002] Gastric cancer is one of the most common high-incidence tumors in my country, and its mortality rate is the second leading cause of death in various malignant tumors both in the world and in China (Bray F, et al 2012; 13(8):790-801). 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, chemotherapy is the main clinical treatment. Although this 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 the later stage cannot be ignored and urgently needed. Therefore, identifying molecular markers and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 黄家强吕有勇潘元明林舒晔邢蕊
Owner BEIJING JIAOTONG UNIV
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