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DNA methylation detection method based on quantitative PCR technology and kit thereof

A technology of technical detection and methylation, applied in biochemical equipment and methods, determination/inspection of microorganisms, and analysis of two-dimensional or three-dimensional molecular structures, etc. Accuracy etc.

Pending Publication Date: 2022-01-28
朱运峰
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Problems solved by technology

The principle is to use sodium sulfite to convert unmethylated cytosine into uracil, and then detect it by sequencing or methylation-specific PCR. Due to the large difference in the conversion rate caused by sodium sulfite, there is insufficient conversion, resulting in Subsequent detection results vary greatly, resulting in inaccurate results; high-resolution melting curve method is to judge whether DNA methylation occurs by observing the change of melting curve after PCR amplification is completed, and the gene structure appears due to sodium sulfite treatment. The different heterogeneity of different heterogeneities has a great influence on the judgment of the results, so the analysis of this method is relatively complicated; the high-throughput sequencing detection method first needs to build a library for the detected DNA, not only for the amount of DNA, but also during the library construction process. It will also cause the loss of some information, and other factors include the high cost and long cycle involved; the combination of methylation-sensitive restriction endonuclease treatment and qPCR analysis is affected by the methylation-sensitive restriction endonuclease Restriction of the site of action, that is, if the detection area does not contain this site, it cannot be detected

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  • DNA methylation detection method based on quantitative PCR technology and kit thereof
  • DNA methylation detection method based on quantitative PCR technology and kit thereof
  • DNA methylation detection method based on quantitative PCR technology and kit thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0089] How to set up a methylation detection method

[0090] The study in this example found that compared with unmethylated DNA, the physical and chemical properties of methylated DNA have significant changes. Based on this phenomenon, a new methylation detection method was explored. The basic principle is that under certain conditions, the difference in DNA methylation is converted into the difference in the amount of effective template for PCR, and then the detection is completed by quantitative PCR. In the modeling process, use PCR to prepare different degrees of methylated DNA fragments through base incorporation, and then detect the difference in methylation, and establish the corresponding relationship between the detection value and the degree of methylation. The specific method as follows:

[0091] 1) Experimental materials

[0092] Reagent: Taq enzyme premix system (2×Premix Ex Taq TM ) and Taq TM Reagents were purchased from TaKaRa Bio Inc; 5'-m-dCTP was purchas...

Embodiment 2

[0137] Lung cancer cf / ctDNA methylation detection method

[0138] 1) Identification of lung cancer target genes:

[0139] HOXD12 gene: Through the analysis of the TCGA database, it was found that the methylation of the HOXD12 gene had the largest difference between normal tissues and lung cancer tissues, and random forest statistical analysis model analysis showed that it had the greatest contribution to the identification of lung cancer. HOXD12 is one of the members of the homeobox gene family, which plays an important regulatory role in the process of morphogenesis. Current studies have shown that different members of this family play an important role in the development of tumors.

[0140] Cytosine deaminase gene (AID): Studies have shown that cytosine deaminase turns cytosine into uracil through the deamination of cytosine. Abnormal expression of this gene can lead to various mutations in DNA and is an important factor for genome variation. Factors, studies have shown th...

Embodiment 3

[0175] Detection of cf / ctDNA methylation in rectal cancer

[0176] 1) Identification of colorectal cancer target genes:

[0177] Through the analysis of the TCGA database, it was found that the methylation of the three genes of SDC2 / WDR17 / ADHFE1 had the largest difference between normal tissues and colorectal cancer tissues, and then the random forest statistical model analysis showed that the methylation of the three genes was the most important in the identification of colorectal cancer. Great contribution. Subsequently, the methylation of the promoter regions of the three genes was detected according to the aforementioned method. In addition, the Alu gene fragment was selected as the proxy of the total amount of free DNA in peripheral blood, and its quantitative detection was carried out.

[0178] Table 14 Target gene primer and probe sequences

[0179]

[0180]Note: The PCR fragment length of target gene amplification target region: WDR17 (promoter region): 124bp; SD...

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Abstract

The invention provides a DNA methylation detection method based on a quantitative PCR technology and a kit thereof, and belongs to the technical field of biology. Detection of DNA methylation is realized based on a quantitative PCR technology, methylated DNA fragments of different degrees are prepared by using PCR through basic group doping, then quantitative PCR detection is performed on the methylated DNA fragments, and a corresponding relation between Ct and DNA methylation degrees is established. Therefore, the kit evaluates the DNA methylation degree of the target gene by detecting the Ct of the target gene. The invention establishes a novel and more convenient detection method for detecting DNA epigenetics modification difference. A lung cancer and colorectal cancer blood sample experiment proves that the method is simple and convenient to operate, short in detection time, accurate and reliable in result, suitable for detection of all types of methylated DNA fragments and extremely high in application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for detecting DNA methylation based on quantitative PCR technology and a kit thereof. Background technique [0002] DNA methylation has become an important part of molecular biology research and a new favorite of disease detection. Epigenetic research has found that DNA epigenetic modification plays an important role in the process of gene regulation, and gene regulation is largely caused by gene epigenetic modification, among which DNA methylation is a hotspot in the study of epigenetic modification. For a long time, the study of gene abnormalities has mainly focused on their structural changes, but the structural changes of genes are theoretically located after the abnormal regulation of gene epigenetics, and the location and frequency of their occurrence are highly uncertain. More sporadic. In addition, if the proportion of DNA with abnormal genetic structur...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/6886G16B30/00G16B15/30
CPCC12Q1/6851C12Q1/6886G16B30/00G16B15/30C12Q2600/154C12Q2531/113C12Q2537/164C12Q2537/165C12Q2545/114C12Q1/686C12Q1/68
Inventor 朱运峰刘天懿高旭东
Owner 朱运峰
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