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Detection kit and method for DNA methylation

A detection method and methylation technology, applied in the field of analysis, can solve the problems of quantitative error of absolute value of methylation level, no cost-benefit advantage, inability to detect methylation levels in regions other than CpG islands, etc., and achieve increased annealing. effect of temperature

Inactive Publication Date: 2019-02-26
THE SIXTH AFFILIATED HOSPITAL OF SUN YAT SEN UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (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
[0005] (2) Unable to detect methylation levels in regions other than CpG islands
If the production batches of methylation standard products are different or there are inevitable quality defects, such as the CG site is not 100% methylated, m5C spontaneously deaminated into C, DNA degradation, etc., will result in the quantification of the absolute value of the methylation level serious error
[0007] The current methylation detection is aimed at the detection of the methylation site of the CpG island, unless sequencing (bisulfite pyrosequencing) is used, such a site can be detected, but it cannot be used in large-sample cohort verification. cost-effective

Method used

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  • Detection kit and method for DNA methylation
  • Detection kit and method for DNA methylation
  • Detection kit and method for DNA methylation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0119] Example 1 Quantitative method for DNA methylation at CpG sites

[0120] DNA isolation and bisulfite conversion

[0121] 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].

[0122] QASM detection

[0123] After bisulfite conversion, real-time fluorescent quantitative PCR was performed to amplify the genomic DNA. Briefly, bisulfite-converted genomic DNA is amplified using primers and a pair of oligonucleotide probes covering the CpG sites to be tested, each oligonucleotide probe linked to a fluorescent reporter at the 5' end Dye 6FAM or VIC (specifically binding to CG sequence and TG sequence, respectively), 3' end coupling quencher-MGB group (MGB-NFQ) ( figure 1 ). Upon DNA extension, the 5' to 3' exonuclease activity of Taq DNA polymerase cleaves the probe and releases the reporter gene, whose fluoresce...

Embodiment 2

[0151] Example 2 Bisulfite Pyrosequencing Comparative Evaluation of Quantitative Accuracy

[0152] Using pyrosequencing as a reference, the accuracy of the methylation quantification method 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, the present technique has the same quantitative accuracy as bisulfite pyrosequencing, but is simpler, more accessible, and less expensive.

Embodiment 3

[0153] Example 3 Comparison of repeatability with techniques using Alu-C4 reaction correction

[0154] In the past, all qPCR-based detection techniques required internal reference genes for quantification. Traditional MethylLight widely uses AluC4 as a control reaction to assess and correct the total amount of starting DNA template [8,11,12]. This high-copy control amplicon is less susceptible to cancer-associated gene alterations than other single-copy control genes. Unfortunately, the effects of genome-wide copy number variation and mutations on AluC4 stability still cannot be completely avoided. The advantage of the present invention is that without such internal reference reaction, accurate PMR can be obtained for each independent sample regardless of the total amount of starting DNA template. In addition, we compared the reproducibility of these two detection methods in 10 clinical tumor samples (the specific steps of traditional MethyLight refer to Eads CA, Danenberg K...

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Abstract

The invention relates to a detection kit and method for DNA methylation, especially to a detection kit and method for DNA methylation without CpG sites at flanking sequences. The kit and method employa Taqman probe binding to a minor groove binder (MGB) and can determine the methylation of both a CpG island (CGI) and isolated CpG sites in CpG open sea and the like without CpG at two flanking sequences. The kit and method provided by the invention are specific and sensitive, simple to operate, and does not require a control reaction or a fully-methylated standard DNA substance as a reference,thereby overcoming defects resulting from the control reaction and the fully-methylated standard DNA substance. In addition, the kit and method have higher repeatability and accuracy than the existingMethyLight technology.

Description

technical field [0001] The invention belongs to the technical field of analysis methods, and relates to a DNA methylation detection kit and method, in particular to a DNA methylation detection kit and method without CpG sites for flanking sequences. 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]. However, 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 methylation of this region Function is unclear. Recent studies have revealed a synergistic effect of methylation...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12M1/34
CPCC12Q1/6858C12Q2523/125C12Q2531/113C12Q2561/101
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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