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Tumor marker STAMP-EP3 based on methylated modification

A methylation and tumor technology, applied in recombinant DNA technology, biochemical equipment and methods, and microbial determination/examination, etc., can solve the problem of insufficient sensitivity and specificity, misdiagnosis, affecting the sensitivity and accuracy of markers And other issues

Active Publication Date: 2019-04-02
SHANGHAI EPIPROBE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are many deficiencies in this technique: first, the sensitivity and specificity are not high enough, the tumor itself has great heterogeneity, including cell populations of various subtypes, and the proportion of tumor DNA in clinical samples, especially blood samples, is very small , the existing tumor markers are difficult to meet the sensitivity of clinical requirements, and it is easy to cause misdiagnosis in the clinic; secondly, a marker only has a good effect on one or a few types of tumors, and the source of DNA in blood is very complicated. Therefore, the existing tumor markers cannot deal with complex issues such as tumor origin and metastasis
Due to the existence of these complex situations, it is difficult to have a uniform standard of use for many DNA methylation tumor markers in clinical application, which seriously affects the sensitivity and accuracy of the markers.

Method used

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  • Tumor marker STAMP-EP3 based on methylated modification
  • Tumor marker STAMP-EP3 based on methylated modification
  • Tumor marker STAMP-EP3 based on methylated modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1. Nucleic acid sequences detected for STAMP-EP3

[0074] The sequence of STAMP-EP3 tumor marker is provided, as shown in SEQ ID NO: 1 below (chr4: 41882451-41882922, Human / hg19), wherein the underlined base is a methylated CpG site, and the number below the underline indicates this position point number.

[0075]

[0076]

[0077] The reverse complementary sequence of the nucleotide sequence shown in above-mentioned SEQ ID NO:1 is as follows SEQ ID NO:3:

[0078] CG GTCAGATTA CG CTCACTAACACC CG AGCTTGTTATCTGACCTGGCCATCCC CG TCACATTCTTTCTACAAGTTATCTTTTCTCCAACCAGGGTATTTGGAGATTATT CG CACTGAATTTTCTGCC CG CCAAGAA CG AATAGGATTGCCAAGCCACACCACTTTTTGGAGCC CG CTTATT CGCG CCTATCCACCCTCTCCTGTGCCCCAGGTTCCCTGAGCA CG GGAATCCTTTC CG GGCATGGCCAAGTTTGTT CG GTGGCTCAAGAG CG GGAAGGGAAGTGCAGTT CG ACACCTGTCCAGCTGCTC CG CTTGGAGATCAAAGGC CG GCTATGGGCTGAG CG ACAGATTTA CG GGA CG GTGGTACAGATTAAGG CG AGAACCCTGC CG GTCCTGGACT CG AGTT CG CA...

Embodiment 2

[0081] Example 2. Methylation differences between STAMP-EP3CpG sites in tumor cells and non-tumor cells—Sequencing after bisulfite treatment (BSP-Bisulfite Sequencing PCR)

[0082] 1. Extract genomic DNA from liver cancer cell line HepG2 and normal liver cell line;

[0083] 2. Treat the extracted HepG2 and normal liver cell line genomic DNA with bisulfite, respectively, as the template for subsequent PCR amplification; in this experiment, the EZ DNA Methylation-Gold Kit from ZYMO Research, article number D5006 is used; but the present invention does not. limited to this kit;

[0084] 3. Design amplification primers (SEQ ID NOs: 5-6; Table 1) according to the sequence of SEQ ID NO: 1, and carry out amplification by conventional methods;

[0085] 4. After PCR amplification, 2% agarose gel electrophoresis was used to detect the specificity of the PCR fragments, the gel was cut to recover the target fragments, ligated and inserted into the T vector, transformed into competent E. ...

Embodiment 3

[0089] Example 3. Methylation differences between STAMP-EP3CpG sites in tumor cells and non-tumor cells—pyrosequencing

[0090] 1. Obtain clinical samples: obtain para-cancer / non-cancer-cancer tissue samples from the clinic, the para-cancer / non-cancer samples are used as the control group, and the cancer tissue samples are used as the tumor detection experimental group;

[0091] 2. DNA extraction: extract the DNA of the experimental group and the control group respectively; this experiment uses the phenol-chloroform extraction method, but the present invention is not limited to this method;

[0092] 3. Bisulfite treatment: Treat the extracted DNA samples with bisulfite, and operate in strict accordance with the steps; in this experiment, the EZ DNA Methylation-Gold Kit from ZYMO Research, product number D5006 was used, but the present invention is not limited to this kit ;

[0093] 4. Primer design: According to the characteristics of STAMP-EP3 sequence SEQ ID NO: 1, PCR ampl...

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Abstract

The invention provides a methylated tumor marker STAMP-EP3 and an application thereof, and belongs to the field of disease diagnosis markers. The invention provides the application of the methylated tumor marker STAMP-EP3 in preparation of cancer diagnostic reagents. The tumor marker STAMP-EP3 is hypermethylated in all types of tumors and hypomethylated in corresponding normal tissues, and has quite high sensitivity and specificity. Primers for detecting the STAMP-EP3 can be used for preparing a tumor diagnosis kit.

Description

technical field [0001] The present invention belongs to the field of disease diagnostic markers, and more particularly, the present invention relates to a methylation-modified tumor marker STAMP (Specific Tumor Aligned Methylation of Pan-cancer). Background technique [0002] The notion that tumors are considered an inherited disease has persisted in the art for decades. Several systematic large-scale sequencing in humans has confirmed that the number of somatic mutations in cancer tissue is significantly lower than expected, and these results suggest that cancer is not simply a hereditary disease. [0003] In order to realize the diagnosis of tumors, many new tumor markers have been discovered and used in clinical diagnosis in recent years. Before 1980, tumor markers were mainly some hormones, enzymes, proteins and other cell secretions, such as carcinoembryonic antigen (CEA), alpha-fetal antigen (AFP), etc., which can be used as markers for gastric cancer and liver cancer...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/154C12Q1/6806C12Q1/6811
Inventor 李振艳罗怀兵
Owner SHANGHAI EPIPROBE BIOTECH CO LTD
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