Probe set for diagnosing gene methylation and its application

A technology of methylation and probe set, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., and can solve the problem that bisulfite modified products cannot be paired and non-methylated probes Problems such as pairing and ineffective detection

Active Publication Date: 2016-08-24
GENE TECH SHANGHAI COMPANY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to the complexity of biological samples, incomplete methylation exists in many samples, and their bisulfite modified products can neither fully pair with methylated probes nor fully pair with unmethylated probes , conventionally designed methylation probes cannot effectively detect such samples

Method used

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  • Probe set for diagnosing gene methylation and its application
  • Probe set for diagnosing gene methylation and its application
  • Probe set for diagnosing gene methylation and its application

Examples

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Effect test

Embodiment 1

[0057] Embodiment 1, the methylation level detection example of MGMT gene

[0058] In this example, methylation probes were designed using the method of the present invention, combined with fluorescent PCR technology, to detect the methylation of the O6-methylguanine-DNA-methyltransferase (MGMT) gene. The MGMT gene is located at 10q26 on chromosome 10, and its function is to remove O6-guanine adducts from DNA and repair damaged DNA. When the CpG site in the promoter region of the MGMT gene is methylated, MGMT transcription stops, and the transfer of the methyl group on guanine-O6 fails, resulting in the inability to effectively repair DNA alkylation damage, which is closely related to tumorigenesis. The results of the study showed that: MGMT promoter methylation is closely related to the clinical effect of the first-line chemotherapy drug Temozolomide. It is generally believed that tumor cells with MGMT methylation are effective against alkylating agents, while non-methylatio...

Embodiment 2

[0086] Embodiment 2, the detection example of three kinds of fluorescent PCR systems to brain glioma sample

[0087] 80 cases of paraffin tissue samples of clinically confirmed glioma were taken, and 5-10um slices were made. Take 3-5 slices into a 1.5mL centrifuge tube, after deparaffinization with xylene, add 180μl DNA extraction solution and 20μl proteinase K solution, digest at 56°C until no visible tissue pieces; add 300μl GDT buffer, mix well after 70 Incubate at ℃ for 10 minutes; add 300 μl absolute ethanol, mix well, transfer to a spin column, centrifuge for 1 minute, discard the filtrate in the collection tube; add 700 μl washing solution PW, centrifuge at 8000 rpm for 1 minute, discard the filtrate; Add 700 μl washing solution WA, centrifuge at 8000 rpm for 1 minute, discard the filtrate; dry the residual liquid in the spin column (centrifuge at 12000 rpm for 2 minutes); add 100 μl DNA eluent to the center of the membrane in the spin column, and place at room temperat...

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Abstract

The invention relates to a probe group for diagnostic gene methylation and application of the probe group, and discloses a preparation method of an optimized gene methylation probe. The optimized methylation detection probe is obtained by introducing a dideoxyinosine nucleotide base to the specific position of a methylation probe. The optimized methylation detection probe can be combined with completely methylated sample DNA, and can tolerate 1-2 non-methylated sites in a binding region, so that the detection efficiency of incompletely methylated samples is effectively improved.

Description

technical field [0001] The invention belongs to the field of gene detection, and more specifically, the invention relates to a probe set for diagnosing gene methylation and its application. Background technique [0002] DNA methylation is one of the ways of gene epigenetic modification, which may exist in all higher organisms. DNA methylation shuts down the activity of certain genes, while demethylation induces reactivation and expression of genes. In mammals, DNA methylation only occurs on CpG cytosine, and under the action of DNA methyltransferases (DNMTs), the cytosine at the 5' end of the CpG dinucleotide is converted into 5' methylcytosine ( m C). This DNA modification does not change the gene sequence, but it can regulate gene expression. DNA methylation is the most well-studied epigenetic mechanism, which plays an important role in the maintenance of normal cell function, modification of chromatin structure, X chromosome inactivation, genomic imprinting, embryonic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12N15/10
CPCC12Q1/6876C12Q2549/00
Inventor 王东李宾梁旺
Owner GENE TECH SHANGHAI COMPANY
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