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Gene methylation detection method

A detection method and methylation technology, applied in the field of genetic engineering, can solve the problems of false positives, cumbersome operation steps, long time-consuming, etc., and achieve the effects of strong specificity, simple operation and high sensitivity

Pending Publication Date: 2018-06-01
HUNAN YEARTH BIOTECHNOLOGICAL CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection of gene methylation is mainly through bisulfite treatment, and then differentiated by methylation-specific PCR or sequencing methods, which are prone to false positives caused by incomplete conversion of bisulfite As a result, the operation steps are cumbersome, many types of reagents are required, and it takes a long time

Method used

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  • Gene methylation detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] In this example, the methylation of the target gene Septin9 gene is detected, and the materials involved are:

[0030] Plasma samples to be tested were mixed with methylated and non-methylated standard DNA (Human Methylated&Non-Methylated DNA Set, Catalog#D5014, ZYMO RESEARCH), the proportion of methylated DNA was 1% and 0%, respectively, and the concentration Both were 2ng / μL.

[0031] Instruments: Roche Lightcycler 480II real-time fluorescent quantitative PCR instrument, ordinary PCR instrument, spiral mixer, water bath, vortex oscillator.

[0032] Specifically follow the steps below:

[0033] (1) In this example, the sample to be tested is normal human plasma or plasma from patients with colorectal cancer, and the DNA of the sample to be tested is extracted using a kit (TheMagMAXTM Cell-Free DNA Isolation Kit). For specific operations, refer to the product manual of the kit; obtain the sample After DNA, use Qubit3.0 to measure the concentration, the qualified sampl...

Embodiment 2

[0057] see Figure 2-5 , this embodiment is an experiment of the minimum nucleic acid content and minimum detection limit required by the method of the present invention, still taking the Septin9 gene as an example.

[0058] In this example, methylated and non-methylated standard DNA (Human Methylated & Non-Methylated DNA Set, Catalog#D5014, ZYMO RESEARCH) was used to blend different sample starting amounts, and the proportion of methylated DNA was 1%, as shown in the table 3 shows:

[0059] table 3

[0060] sample number

[0061] Use methylated and non-methylated standard DNA (Human Methylated&Non-Methylated DNASet, Catalog#D5014, ZYMO RESEARCH) to mix different proportions of methylated DNA, the initial amount is 2ng, as shown in Table 4:

[0062] Table 4

[0063] sample number

[0064] Take 10U of restriction endonuclease AciI to digest the samples a1, a2, a3, a4, a5, a6 prepared above; See Example 1) for the system, amplification procedure, primers ...

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Abstract

The invention belongs to the technical field of gene engineering and in particular relates to a gene methylation detection method. According to the gene methylation detection method provided by the invention, nucleic acid to be detected is treated with a restriction enzyme, and in addition, specific primer probes are designed for nucleotide sequences in an area to be detected, and PCR (PolymeraseChain Reaction) amplification and fluorescent signal detection can be implemented. The gene methylation detection method has the advantages of being simple to operate, good in specificity and high insensitivity.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for detecting gene methylation. Background technique [0002] DNA methylation is one of the earliest methods of gene epigenetic modification. Methylation in eukaryotes only occurs on cytosine, that is, under the action of DNA methyltransferase, the CpG dinucleotide 5'- end cytosine is converted to 5'-methylcytosine. [0003] DNA methylation usually inhibits gene expression. For example, when a tumor occurs, the degree of unmethylation of the CpG sequence outside the CpG island of the tumor suppressor gene increases, and the CpG in the CpG island is highly methylated, resulting in a decrease in the expression of the tumor suppressor gene. decline. Therefore, studying the methylation of genes is of great significance to the early screening of cancer. [0004] At present, the detection of gene methylation is mainly through bisulfite treatment, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12Q1/6851
CPCC12Q1/6851C12Q1/6858C12Q2521/301C12Q2531/113C12Q2563/107C12Q2545/101
Inventor 秦闯华陆利徐根明潘艺赵谦
Owner HUNAN YEARTH BIOTECHNOLOGICAL CO LTD