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Primer capable of detecting methylation of promoter of gene related to colonic adenocarcinoma

A methylation and gene technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of limited practical application of detection instruments, cumbersome operation, low sensitivity, etc., to avoid Intra-chain and inter-chain complementarity issues, ensuring specificity, and reducing the effect of background interference

Active Publication Date: 2013-03-06
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional DNA sequencing method is cumbersome to operate and has low sensitivity; although Real time PCR has high sensitivity, its cost is high. In addition, the demand for detection equipment also limits its practical application in clinical practice.

Method used

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  • Primer capable of detecting methylation of promoter of gene related to colonic adenocarcinoma
  • Primer capable of detecting methylation of promoter of gene related to colonic adenocarcinoma
  • Primer capable of detecting methylation of promoter of gene related to colonic adenocarcinoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Embodiment 1, the design of primer

[0084] 1. Selection of candidate genes

[0085] Selected candidate genes with high frequency of methylation associated with colon adenocarcinoma, selected VIM (NM_003380), MGMT (XM_003483574), TMEFF2 / HPP1 (NM_016192), ESR1 (NM_000125), APC (NIM_001127511), and MLH1 (NM_000249) gene sequence. The sequences of these genes, including all exons and introns, can be found through the link http: / / www.ensembl.org. The positions of the amplified sequences are as follows:

[0086] serial number

Gene

Location

starting base position

NM_003380

VIM

exon 3

-75～+105

XM_003483574

MGMT

first exon

-370～-224

NM_016192

TMEFF2 / HPP1

first exon

-385～-213

NM_000125

ESR1

exon 6

+62～+226

+NIM_001127511

APCs

first exon

-140～-86

NM_000249

MLH1

first exon

-500～-207

[0087] 2. Design primers

[0088] Find...

Embodiment 2

[0093] Embodiment 2, the application of primer

[0094] 1. Application of VIM primers

[0095] The efficiencies of these primers were verified on DNA samples derived from colon adenocarcinoma, adenoma, and normal human tissue samples according to the CCP-FRET detection method reported in references. Specific process:

[0096] 1. Synthesis of PFP

[0097] PFP was synthesized according to the method reported in the reference (Liu, B., Wang, S., Bazan, G.C. & Mikhaillovsky,, A Shape-adaptablewater-soluble conjugated polymers.J.Am.Chem.Soc.2003, 125, 13306-13307) (the structural formula is as above).

[0098] 2. Extraction of DNA to be tested

[0099] Genomic DNA was extracted from 30 colon adenocarcinomas, 30 colon adenomas, and 50 normal human tissue samples (ex vivo blood, from the First Affiliated Hospital of the General Hospital of the Chinese People’s Liberation Army, where the patients were informed and diagnosed).

[0100] The DNA samples obtained above were digested ...

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Abstract

The invention discloses a primer capable of detecting the methylation of a promoter of a gene related to colonic adenocarcinoma. The primer which is used for detecting the methylation of a DNA (Deoxyribonucleic Acid) to be detected provided by the invention consists of a primer pair A, a primer pair B, a primer pair C, a primer pair D, a primer pair E and a primer pair F, wherein the primer pair A consists of a primer 1 and a primer 2, the primer pair B consists of a primer 3 and a primer 4, the primer pair C consists of a primer 5 and a primer 6, the primer pair D consists of a primer 7 and a primer 8, the primer pair E consists of a primer 9 and a primer 10, the primer pair F consists of a primer 11 and a primer 12, and nucleotide sequences of the primers 1-12 are sequences 1-12 in the sequence table. When the primer capable of detecting the methylation of the DNA is adopted, a Tm value of the primer can be controlled at 60 DEG C, specificity and amplification efficiency of a PCR (Polymerase Chain Reaction) product are ensured, and methylated state of samples of colonic adenocarcinoma and adenoma can be efficiently detected.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a series of primers for detecting the methylation of colon adenocarcinoma-related gene promoters. Background technique [0002] The methylation modification of the promoter CpG region of tumor-related specific genes is related to tumorigenesis and formation. It is worth noting that different types of tumors and different pathological types and grades of the same tumor will have specific gene methylation modifications. Therefore, the methylation status of tumor-related genes has become an important class of tumor detection markers , can be used for early detection of tumors, evaluation of tumor prognosis and prediction and evaluation of drug sensitivity. [0003] Conjugated polymer-based fluorescence resonance energy transfer (Cationic conjugated polymer-based fluorescence resonance energy transfer, CCP-FRET) can improve the sensitivity and specificity of detection. Applying this tec...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 王树杨琼吴蔚
Owner INST OF CHEM CHINESE ACAD OF SCI
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