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Method of simultaneously testing multiple gene DNA methylation and application thereof

A methylation and multi-gene technology, applied in the field of design detection, can solve problems such as difficulty, low throughput, and inability to perform multi-gene detection, and achieve easy-to-interpret, high-throughput, and high-specificity effects

Inactive Publication Date: 2010-05-05
SHANGHAI CHEST HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many methods for detecting DNA methylation at specific sites, such as MS-PCR, MS-DHPLC, sequencing, etc., but the throughput of these methods is low, and it is difficult to detect the degree of methylation of multiple genes at the same time Large, not even possible for polygenic testing

Method used

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  • Method of simultaneously testing multiple gene DNA methylation and application thereof
  • Method of simultaneously testing multiple gene DNA methylation and application thereof
  • Method of simultaneously testing multiple gene DNA methylation and application thereof

Examples

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Embodiment 1

[0043] Embodiment 1 Simultaneous detection of human p16 INK4a Preparation of kits for DNA methylation of seven genes, RASSF1A, MGMT, DAPK, RARβ, GSTP1, and APC

[0044] (1) Design and synthesis of gene junction probes and universal primer sequences.

[0045] Design connection probes and universal primers according to the following principles: A) Each gene needs two probes, which are called upstream probes and downstream probes according to their 5'-3' order, and the upstream probes are from 5' to 3' They are the universal primer sequence, the address sequence (each gene is different, and it is used to identify different genes when used in liquid chip detection) and the corresponding gene-specific sequence, and the downstream probes are the corresponding gene-specific sequence and universal sequence from 5' to 3' respectively. Primer sequence; B) both the upstream and downstream probes are complementary to the same strand of the DNA to be tested, and the downstream probe is fo...

Embodiment 2

[0060] The use of embodiment 2 kit

[0061] (1) Experimental method:

[0062] (1) Take 500ng of genomic DNA extracted from the surgically resected tissues of 8 lung cancer patients, and use Qiagen's Bisulfite kit to treat unmethylated C in the DNA to U, and the obtained sample is used for testing.

[0063] (2) Ligation reaction

[0064] Add 1 μL of sample as a template, 2 μL of upstream and downstream probes (1 μM for each probe in Table 1), 0.8 μL hybridization buffer (Tris-HCl 300 mM, 1.5M KCl, 1 mM EDTA, pH 8.5), 4.2 μL ddH 2 After O mixing, first 95°C for 5 minutes, then annealed at 55°C to 70°C for 60 minutes; then add 12 μL ligation system (containing 0.5 μL Taq DNA ligase and 2 μL corresponding enzyme buffer, 9.5 μL ddH 2 O) Into each hybridization reaction system, react at 54°C for 15min after mixing, and then inactivate the enzyme at 98°C for 10min;

[0065] (3) PCR reaction

[0066] Take 4 μL of the ligation product and add it to 16 μL of the PCR reaction master ...

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Abstract

The invention discloses a method of simultaneously testing multiple gene DNA methylation and an application thereof. The method comprises the following steps: firstly using the sulfite method to treat a DNA to be tested, then modifying treated DNA to hybridize with a connecting probe, then performing a ligation under the action of DNA ligase, using a general primer to perform PCR amplification, mixing the product with fluorescent coding microspheres crosslinked with various inverse bar codes hybrid probes and reading analysis data with a liquid chip platform. The invention further discloses a kit designed through the method of the invention. The method of simultaneously testing multiple gene DNA methylation can be used for testing DNA methylation in high flux.

Description

technical field [0001] The invention is in the field of design detection, and more specifically relates to a detection method and application of polygenic DNA methylation. Background technique [0002] The DNA of various organisms is modified by methylation, which has the function of regulating gene expression. The DNA methylation modification of higher organisms including humans is mainly the methylation of C (cytosine) in the CpG sequence. In mammals, the frequency of CpG sequences in the genome is only 1%, much lower than other dinucleotide sequences in the genome. However, in some regions of the genome, the CpG sequence density is very high, which can reach more than 5 times the average value, and become the enriched region of guanine and cytosine, forming the so-called CpG island, usually located in the promoter region of the gene or the first an exon region. If the CpG island of the DNA of a gene is heavily methylated, the expression of the gene will be inhibited. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 娄加陶
Owner SHANGHAI CHEST HOSPITAL
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