Method for quantitative PCR detection of maspin gene methylation state and application thereof

A methylation and non-methylation technology, which is applied in the fields of biotechnology and medicine, can solve problems such as difficult to detect, inability to detect early in situ breast cancer, and inability to meet tumor molecular typing, so as to avoid accidental errors, Optimizing the PCR system and procedures to ensure the effect of accuracy

Inactive Publication Date: 2019-01-18
WUHAN UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (1) Cannot detect the occurrence of early breast cancer in situ, that is, it is difficult to detect when there is no obvious tumor or the tumor mass is small in the early stage of breast cancer
[0005] (2) It is difficult to predict whether breast cancer will metastasize
[0

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  • Method for quantitative PCR detection of maspin gene methylation state and application thereof
  • Method for quantitative PCR detection of maspin gene methylation state and application thereof
  • Method for quantitative PCR detection of maspin gene methylation state and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Determine the specific site and sequence information of the CpG island in the upstream regulatory region of the maspin gene coding sequence

[0046] The breast cancer detection method of the present invention is based on the principle of methylation-specific PCR. After the single-stranded DNA is modified by bisulfite, all unmethylated cytosines are deaminated into uracils, and the methyl groups in the CpG sites are The modified cytosine remains unchanged, and the formazan can be amplified by PCR

[0047] Differentiate between methylated and unmethylated DNA sequences. Therefore, it is necessary to first determine the specific site and sequence information of the CpG island in the upstream regulatory region of the maspin gene coding sequence.

[0048] Use the gene sorter tool at http: / / genome.ucsc.edu / index.html, select the species as human, select the latest database, search for the maspin gene, select the upstream 2000bp and 5'UTR of the promoter, and obtai...

Embodiment 2

[0049] Example 2: Design and synthesis of primers for specifically amplifying the CpG island of the upstream regulatory region of the maspin gene coding sequence

[0050] According to the CpG island information of the upstream regulatory region of the maspin gene coding sequence in Example 1, two sets of primers for methylated and unmethylated sequences were designed, and these 10 pairs of primers can be used to specifically amplify the upstream regulation of the maspin coding sequence The region contains methylated and unmethylated sequences of different lengths of CpG islands, and subsequent methylation quantitative analysis is performed to specifically detect the methylation status of the corresponding CPG site. According to the sequence shown in SEQ ID NO.1, go through the http: / / www.urogene.org / cgi-bin / methprimer / methprimer.cgi website, select the Pick MSP primers option, and check Use CpG island prediction for primer selection? Then click submit to get the primers used t...

Embodiment 3

[0051] Embodiment 3: establish the method for detecting the methylation state of maspin gene based on quantitative PCR

[0052] The detection method of the present invention is based on the principle of methylation-specific PCR, and primers for methylated and unmethylated sequences are designed respectively, and methylated and unmethylated DNA sequences can be distinguished through PCR amplification . The method comprises the steps of:

[0053] 1. Extract genomic DNA from tissue samples and perform quality inspection on the extracted DNA samples

[0054] According to the diagnostic needs of whether the patient has breast cancer metastasis and malignancy, the tissue sample can be a small amount of breast cancer clinical tissue samples taken out minimally invasively, or it can be a breast cancer tissue sample from the metastatic site of breast cancer; for normal human breast cancer For the needs of early screening such as morbidity, tissue samples can be a small amount of brea...

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Abstract

The invention relates to a method for quantitative PCR detection of maspin gene methylation state and application thereof. The method comprises: firstly, the specific site and sequence information ofCpG island in the upstream regulatory region of maspin gene coding sequence were determined, and 10 pairs of specific primers were designed to detect the methylation status of CpG island in the upstream regulatory region of maspin gene coding sequence. The methylation of maspin was quantitatively detected by using the different length of CpG island in the upstream regulatory region of maspin coding sequence amplified by the primer. CpG island methylation status in breast canc tissue or upstream regulatory region of maspin coding sequence of breast canc metastasis site of breast cancer patientcan be detected by that method, whether breast canc metastasis and malignant degree of the patient have occurred or not can be diagnosed, and the staging classification and treatment of breast cancercan be assisted in clinic; It can also detect the methylation status of CpG island in the upstream regulatory region of maspin coding sequence of normal breast, judge the incidence of breast cancer, metastasis and malignant degree of breast cancer, and assist early screening of breast cancer, so it has a good application prospect.

Description

technical field [0001] The invention relates to the fields of biotechnology and medicine, in particular to a method for detecting the methylation state of maspin gene based on quantitative PCR and its application. Background technique [0002] The International Agency for Research on Cancer 2012 provides estimates of cancer incidence, mortality, and prevalence worldwide, as well as national and regional data. This big data shows that breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women worldwide, with an estimated 1.7 million cases and 521,900 deaths in 2012. Breast cancer alone accounts for 25% of all cancer cases and 15% of all cancer deaths. According to the latest survey report released by the Ministry of Health, in my country, breast cancer has become one of the fastest-rising malignant tumors. In the past 30 years, the increase rate was as high as 96%, second only to lung cancer. In Shanghai, where the situation is most ...

Claims

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Application Information

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IPC IPC(8): C12N15/117C12Q1/6886C12Q1/6858C12N15/11
CPCC12N15/117C12Q1/6858C12Q1/6886C12Q2600/154C12N2310/17
Inventor 廖兴华李佳蓬张慧敏项园李含含黄凤戴周彤张子健王君李会李慧张晓宇张同存
Owner WUHAN UNIV OF SCI & TECH
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