A dna methylation quantitative system

A methylation and non-methylation technology, applied in the field of analysis, can solve the problems of inability to accurately detect genomic heterozygous methylation, inability to detect methylation levels in regions other than CpG islands, and inability to successfully design primers and probes. , to achieve the effect of increasing the annealing temperature

Active Publication Date: 2019-12-24
THE SIXTH AFFILIATED HOSPITAL OF SUN YAT SEN UNIV +1
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] (1) Unable to accurately detect heterozygous methylation present in the genome
If adjacent CG sites do not exhibit co-methylation, primers and probes required for qMSP techniques cannot be successfully designed in this region
[0007] (2) Unable to detect methylation levels in regions other than CpG islands
The regions other than CpG islands in the genome include opensea, CpG shore and other regions, mainly located in the gene body and intergenic regions. There are few CG sites in this region, and there are few adjacent CG sites. It is impossible to design qMSP technology in this region. primers and probes
[0008] Methylation standards may not be required unless sequencing is used (bisulfite pyrosequencing), and non-CpG-to-CpG sites can be detected, but it is not cost-effective in large sample cohort validation

Method used

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  • A dna methylation quantitative system
  • A dna methylation quantitative system
  • A dna methylation quantitative system

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Embodiment 1 A kind of DNA methylation quantitative system

[0103] A DNA methylation quantitative system comprising:

[0104] a) Collection component: used to collect the amount of methylation and the amount of non-methylation of the DNA sample to be tested;

[0105] Specifically, the collection component also includes:

[0106] 1) conversion module: the conversion module is used to convert the unmethylated cytosine base of the DNA sample into uracil, while the methylated cytosine base remains unchanged;

[0107] 2) Amplification module: the amplification module is used to amplify the transformed DNA sample.

[0108] b) Processing components: methylation / (methylation+non-methylation)×100 to calculate the percentage of methylated referenc, PMR.

[0109] As an option, it also contains

[0110] c) Output component: the output component is used to output the processing component to obtain the methylation percentage parameter percentof methylated referenc, PMR.

Embodiment 2

[0111] Example 2 DNA methylation quantification system workflow

[0112] 1. The amount of methylation and non-methylation of DNA samples is collected

[0113] Material

[0114] Fresh-frozen tumor tissue samples were obtained from 45 patients with primary colorectal adenocarcinoma. Patients included 32 men and 13 women. Among these patients, 17 had stage I tumors and 28 had stage II tumors; 18 had relapsed and 27 had no recurrence. See Table 1 for details.

[0115] Table 1 Tumor samples

[0116]

[0117]

[0118] 2. The conversion module converts the unmethylated cytosine bases of the DNA sample to be tested

[0119] Using the QIAamp DNA Mini Kit (Qiagen, 51306) and the EZ DNA Methylation Kit (ZymoResearch, D5002), the genomic DNA in the above samples was extracted and modified with bisulfite according to the instructions [7,14].

[0120] 3. The amplification module amplifies the transformed DNA sample

[0121] After bisulfite conversion, real-time fluorescent quan...

Embodiment 3

[0147] Example 3 Bisulfite Pyrosequencing Comparative Evaluation of Quantitative Accuracy

[0148] Using pyrosequencing as a reference, the accuracy of the methylation quantification system of the present invention was further tested. We used pyrosequencing and this technology to detect the methylation levels of the above three CpG sites in 10 colorectal cancer tissues (Table 1). The primers for pyrosequencing are listed in Table 2. The PMR measured by the present invention is linearly correlated with the methylation percentage obtained by pyrosequencing ( image 3 ; FAT3 was 0.9690, FHIT was 0.9954, KIAA1026 was 0.8755, all P<0.001). Not only that, there is a striking agreement between PMR and Pyrosequencing in terms of percent methylation. This is more precise than traditional MethyLight reported previously [11,12]. Therefore, this system has the same quantitative accuracy as bisulfite pyrosequencing, but is easier to operate, more accessible, and less expensive.

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Abstract

Provided are a DNA methylation quantification system and method. The system comprises an acquisition member for acquiring the quantities of methylated and non-methylated CpGs to be tested in a DNA sample to be tested, and a processing member for calculating percent of methylation (PM) parameters according to methylation / (methylation + non-methylation) × 100%. The system applies not only to the methylation quantification of co-methylated CpG positions in CpG islands, but also particularly to the DNA methylation quantification for flanking sequences having no CpG positions. The system and method do not require a control reaction or a fully-methylated DNA standard as a reference, and thus can overcome defects resulting therefrom, and has higher repeatability and accuracy.

Description

technical field [0001] The invention belongs to the technical field of analysis methods, and relates to a DNA methylation quantitative system. Background technique [0002] Cytosine-5 DNA methylation (m5C) present in cancer tissues is considered as an epigenetic modification of DNA with potential clinical value [1]. In vertebrates, m5C occurs mainly at CpG dinucleotides. Aberrant methylation of CpG islands (CGIs) at the promoters of tumor suppressor genes has been demonstrated in a variety of tumors leading to transcriptional inactivation [2]. The CGI within the promoter represents only a small part of the methylation, while the CpG open sea, mainly located in the gene body, represents the most conserved DNA methylation target in eukaryotes, but the function of methylation in this region is still unknown. Not sure. Recent studies have revealed a synergistic effect of methylation in non-promoter regions (such as gene body and UTR) on gene expression, and gene body methylat...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858
CPCC12Q1/6858C12Q2523/125C12Q2561/101C12Q2563/107
Inventor 禹汇川骆衍新白亮亮唐冠楠王小琳李英杰黄增鸿黄美近汪建平
Owner THE SIXTH AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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