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Primer probe composition for detecting methylation level of DNA (deoxyribonucleic acid) and application of primer probe composition

A primer-probe, composition technology, used in recombinant DNA technology, DNA/RNA fragments, determination/inspection of microorganisms, etc.

Active Publication Date: 2020-06-26
XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A combination of primers and probes has not been disclosed in the prior art for testing METRNL, PDE4D and MAPT gene methylation levels to assist early diagnosis and differential diagnosis of Parkinson's disease

Method used

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  • Primer probe composition for detecting methylation level of DNA (deoxyribonucleic acid) and application of primer probe composition
  • Primer probe composition for detecting methylation level of DNA (deoxyribonucleic acid) and application of primer probe composition
  • Primer probe composition for detecting methylation level of DNA (deoxyribonucleic acid) and application of primer probe composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1 is used to detect the design screening of the primer probe of METRNL, PDE4D and MAPT gene DNA methylation level

[0069] The main principle of the present invention is: in the reaction system of the fluorescent PCR-probe method, a pair of PCR amplification primers and a pair of probes with different fluorescent labels are included. The probe is only specifically combined with the template (ie sample DNA), and the probe binding region is between the primer pair. When the probe is intact, the fluorescent signal emitted by the 5'-end fluorescent group of the probe will be absorbed by the 3'-end quencher group, and the PCR instrument cannot detect the fluorescent signal. With the PCR amplification, the Taq enzyme will When encountering a probe that specifically binds to the template, the 3'-5' exonuclease activity of Taq enzyme will cut off the probe, and the fluorophore will be far away from the quencher group, so that the instrument can detect a specific fluo...

Embodiment 2

[0086] Embodiment 2 A kind of primer probe combination carries out the real-time fluorescent PCR detection method of METRNL, PDE4D and MAPT gene DNA methylation level

[0087] The method for selecting the primer probe combination described in Example 1 to detect the DNA methylation level of METRNL, PDE4D and MAPT genes comprises the following steps:

[0088] 1. Extract the genomic DNA in the sample: Use the blood genomic DNA extraction kit (0.1-1mL) (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., model DP348) to extract the human genomic DNA in the human whole blood sample, and the genomic DNA is subjected to ultraviolet light After spectrophotometer detection, the concentration of genomic DNA is required to be not less than 40ng / μL, OD 260 / OD 280 The ratio is not less than 1.80;

[0089] 2. Sulfite modification of genomic DNA: Add 5.5 μL 3M NaOH solution to 50 μL genomic DNA sample, denature at 42°C for 30 minutes, add 30 μL 10 mM hydroquinone solution,...

experiment example

[0125] Experimental Example The effect of a combination of primers and probes on the detection of DNA methylation levels of METRNL, PDE4D and MAPT genes

[0126] According to the detection method provided in Example 2, 3 different samples were tested, and the sources of samples 1-3 were different peripheral blood DNAs clinically obtained by Xuanwu Hospital.

[0127] The detection results of the methylation level at positions 1-4 of the CpG island of each gene in each sample are as follows:

[0128] Sample 1(%) Sample 2(%) Sample 3(%) METRNL gene CpG island position 1 28 28 29 METRNL gene CpG island position 2 27 25 27 METRNL gene CpG island position 3 29 27 20 PDE4D gene CpG island position 1 33 31 31 PDE4D gene CpG island position 2 40 38 39 PDE4D gene CpG island position 3 26 31 29 PDE4D gene CpG island position 4 34 36 36 MAPT gene CpG island position 1 94 94 94 MAPT gene CpG island position ...

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Abstract

The invention provides a primer probe composition for detecting the methylation level of DNA (deoxyribonucleic acid) and application of the primer probe composition. Nucleotide sequences of primers comprise at least two of SEQ ID NO:1-2, SEQ ID NO:9-10, SEQ ID NO:15-16 and SEQ ID NO:21-22; and nucleotide sequences of probes comprise at least two of SEQ ID NO:3-8, SEQ ID NO:11-20 and SEQ ID NO:23-28. The primer probe composition is used for detecting CpG island methylation levels of METRNL (meteorin like glial cell differentiation regulator), PDE4D (phosphodiesterase 4D) and MAPT (microtubule associated protein tau) genes, furthermore is used for diagnosing patients suffering from Parkinson's diseases, and is particularly applied to differential diagnosis on patients suffering from early-stage Parkinson's diseases and patients suffering from different subtypes of the Parkinson's diseases. The primer probe composition provided by the invention is low in cost, simple in diagnosis operation and high in practical application value.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a set of primer probe compositions for detecting DNA methylation levels and applications thereof. Background technique [0002] The glial cell differentiation regulator-like factor (METRNL) gene is located on human chromosome 17 (NC_000017.11, 83079609-83095126), spanning about 16 kb. METRNL is a homologous protein of the glial cell differentiation regulator Meteorin, and the two have 40% homology in protein sequence. METRNL is involved in various biological processes, such as white fat formation, neurodevelopment, white fat browning, and bone formation. [0003] The phosphodiesterase 4D (PDE4D) gene is located on human chromosome 5 (NC_000005.10), spanning about 1500kb. Studies have shown that the protein encoded by the PDE4D gene has the function of degrading cyclic adenosine monophosphate, and may affect the pathogenesis of ischemic stroke through the process of atherosclerosis....

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/154
Inventor 蔡燕宁张陆明
Owner XUANWU HOSPITAL OF CAPITAL UNIV OF MEDICAL SCI
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