Tumor marker STAMP-EP5 based on methylation modification

A methylation and tumor technology, applied in the direction of recombinant DNA technology, biochemical equipment and methods, microbial measurement/testing, etc., can solve problems such as misdiagnosis, insufficient sensitivity and specificity, difficult to use standards, etc.

Active Publication Date: 2019-03-12
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-EP5 based on methylation modification
  • Tumor marker STAMP-EP5 based on methylation modification
  • Tumor marker STAMP-EP5 based on methylation modification

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1, for the nucleic acid sequence detected by STAMP-EP5

[0073] The sequence of the STAMP-EP5 tumor marker is provided, as shown in the following SEQ ID NO: 1 (chr22: 46658718-46659169 / hg19), where the underline indicates that the base is a methylated CpG site, and the number below the underline indicates the position of the site serial number.

[0074]

[0075] The above sequence of SEQ ID NO:1 is treated with bisulfite and the sequence is as follows: SEQ ID NO:2 (wherein Y represents C or U):

[0076]

[0077]

[0078] The reverse complementary sequence of the nucleotide sequence shown in the above SEQ ID NO:1 is as follows: SEQ ID NO:3:

[0079] CG T CG GC CG CCTCC CG CTGC CG C CG GTTCCT CGCG GGGCACAAGC CG CCGTC

[0080] TC CG GGG CG CC CG GTGGCCTCCTCAGGGG CG CCC CG GGCCT CG GGGTG CGCG

[0081] G CG GC CGCG CCCTCCTCAGTCTG CG GCCCAG CGCG GTG CG GG CG GG CG G CG C

[0082] TGTCCTGAG CG GC CGCG GC...

Embodiment 2

[0098] Example 2. Differences in the methylation of STAMP-EP5CpG sites between tumor cells and non-tumor cells——bisulfite-treated sequencing method (BSP-Bisulfite Sequencing PCR)

[0099] 1. Extract the genomic DNA of the liver cancer cell line HepG2 and the normal liver cell line;

[0100]2. Treat the extracted HepG2 and normal liver cell line genomic DNA with bisulfite, respectively, as templates for subsequent PCR amplification;

[0101] 3. Design amplification primers according to the sequence of SEQ ID NO: 1, design primers (SEQ ID NOs: 5-6; Table 1), and perform amplification by conventional methods.

[0102] 4. After PCR amplification, 2% agarose gel electrophoresis was used to detect the specificity of the PCR fragment, the gel was cut to recover the target fragment, ligated and inserted into the T vector, transformed into competent E. 10 clones were picked for Sanger sequencing.

[0103] Table 1, BSP primers

[0104]

[0105] BSP verification of the methylation ...

Embodiment 3

[0106] Example 3. Differences in the methylation of STAMP-EP5 CpG sites between tumor cells and non-tumor cells—pyrosequencing

[0107] 1. Obtain clinical samples: Obtain para-cancer / non-cancer-cancer tissue samples from the clinic, para-cancer / non-cancer samples are used as the control group, and cancer tissue samples are used as the tumor detection experimental group;

[0108] 2. DNA extraction: extract the DNA of the experimental group and the control group respectively; this experiment uses the phenol-chloroform extraction method, but is not limited to this method;

[0109] 3. Bisulfite treatment: treat the extracted DNA samples with bisulfite, and operate in strict accordance with the steps; in this experiment, EZ DNA Methylation-Gold Kit from ZYMO Research Company, Cat. No. D5006 was used, but not limited to this kit;

[0110] 4. Primer design: According to the characteristics of STAMP-EP3 sequence SEQ ID NO: 1, design PCR amplification primers and pyrosequencing primers...

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Abstract

The invention provides a methylation tumor marker STAMP-EP5 and application thereof and belongs to the field of disease diagnosis markers. The invention provides the application of the methylation tumor marker STAMP-EP5 to preparation of tumor diagnosis reagents. The tumor marker STAMP-EP5 provided by the invention shows high methylation in all tumor types and low methylation in corresponding normal tissues, and has extremely high sensitivity and specificity; a primer for detecting the STAMP-EP5 can be used for preparing a tumor diagnosis kit.

Description

technical field [0001] The invention belongs to the field of disease diagnostic markers, more specifically, the invention relates to a tumor marker STAMP (Specific Tumor Aligned Methylation of Pan-cancer) based on methylation modification. Background technique [0002] The notion that tumors are considered to be a genetic disease persisted in the field for decades. Large-scale sequencing of several human systems has confirmed that the number of somatic mutations in cancer tissues is significantly less than expected, and these results imply that cancer is not a simple genetic 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 cell secretions such as hormones, enzymes, and proteins, such as carcinoembryonic antigen (CEA), alpha-fetoantigen (AFP), etc., which can be used as markers for various tumors such as gastric cancer and liver can...

Claims

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Application Information

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