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Methylated EphA7 nucleotide fragment and detection method and application thereof

A detection method and methylation technology, applied in the fields of genetic engineering and medical detection, can solve the problems of less research on EphA7 and no correlation with cervical cancer, and achieve the effect of avoiding waste

Active Publication Date: 2019-06-04
TIANJIN MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, previous studies mostly focused on its physiological role. In recent years, studies have found that some Eph family genes are related to the occurrence of tumors, but there are relatively few studies on EphA7, and there are no reports on its correlation with cervical cancer.

Method used

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  • Methylated EphA7 nucleotide fragment and detection method and application thereof
  • Methylated EphA7 nucleotide fragment and detection method and application thereof
  • Methylated EphA7 nucleotide fragment and detection method and application thereof

Examples

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Embodiment 1

[0072] Detection of cervical cancer-specific EphA7 gene methylation by pyrosequencing

[0073] 2.1 Pyrosequencing

[0074]The pyrosequencing method is called the gold standard of methylation analysis. Its sequencing principle is as follows: first, sulfite-treated BS-DNA is used, and the gene fragment is amplified by PCR as the template to be sequenced, and then the sequencing part is composed of four enzymes. , including DNA polymerase (DNA polymerase), ATP sulfurylase (ATP sulfurytase), luciferase (luciferase) and ATPase (Apyrase) catalyzed enzyme cascade chemiluminescent reaction in the same reaction system. Only one deoxynucleotide triphosphate (dNTP) is added to each round of sequencing reaction system. If the dNTP is paired with the template, it will be added to the 3' end of the sequencing primer under the action of DNA polymerase, and a molecule of pyrophosphate (PPi) will be released at the same time. Added dNTPs and released PPi are equal amounts. ATP sulfurylase c...

Embodiment 2

[0097] Detection of cervical cancer-specific EphA7 gene methylation by fluorescence quantification

[0098] 3.1 Fluorescent quantitative methylation-specific PCR (QMSP)

[0099] The principle of fluorescent quantitative methylation-specific PCR is that after sulfite treatment of EphA7 DNA, the C base of the methylated CpG site still remains a C base, while the C base of an unmethylated CpG site The base has become a U base, and then converted into a T base in the PCR reaction. Therefore, primers and probes are designed for the specific methylated EphA7 gene fragment (shown in SEQ ID NO: 1), and after QMSP Amplification, only the methylated EphA7 gene is amplified, and unmethylated fragments cannot be amplified.

[0100] 3.2 Primer and probe design

[0101] QMSP primers were designed using methyl-primer v1.0 and synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The sequences are shown in Table 4.

[0102] Table 4 Primer / probe sequence and PCR amplification product si...

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Abstract

The invention relates to the technical fields of genetic engineering and medical detection, and provides a methylated EphA7 nucleotide fragment as a specific diagnosis marker of cervical cancer, and aprimer set, a probe and a reagent kit relevant with the methylated EphA7 nucleotide fragment, and an application of the methylated EphA7 nucleotide fragment to preparation of a cervical cancer screening and diagnosing preparation. The inventor finds that the methylation degree of a promoter region of EphA7 genes in cervical tissues is in significant correlation with the generation of the cervicalcancer, and the methylated EphA7 nucleotide fragment can be used as a biology marker for assisting in diagnosis of the cervical cancer, so that the human cervical cancer can be timely screened and diagnosed for early stage, and the waste of medical resources is avoided.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and medical detection, and in particular relates to a methylated EphA7 nucleotide fragment and a detection method and application thereof. Background technique [0002] Cervical cancer (CC) is one of the most common cancers in women. According to statistics, there were about 570,000 new cases and 311,000 deaths worldwide in 2018. Cervical cancer ranks fourth among female cancers in developed countries, while cervical cancer still ranks second among female cancers in developing countries. The annual number of new cases in my country is more than 133,000, and about 20,000 to 30,000 women die of cervical cancer every year. In recent years, the incidence and mortality of cervical cancer among young women in my country are on the rise. [0003] Cervical cancer is different from other tumors. The natural history of the disease is clear, and there are a series of precancerous lesions. Its o...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11C12Q1/6869C12Q1/6806
Inventor 王蓉曹会玲范永娟赵敬梁政时淑娟
Owner TIANJIN MEDICAL UNIV
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