Detection method of targeting membrane protein methylation as imprinting gene syndrome marker

A detection method, methylation technology, applied in the field of medical detection, can solve the problem of membrane protein SLC22A18 function inhibition, etc., and achieve the effect of cost reduction and high sensitivity

Pending Publication Date: 2022-08-09
SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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Problems solved by technology

[0004] After in-depth research by our team, the imprinted gene SLC22A18-related children's "allergy-short stature-fatty liver" syndrome was defined for the first time. The peripheral blood cells of the children's SLC22A18-DNA promoter and CpG island hypermethylation, suggesting imprinted genetic SLC22A18 hypermethylation It affects the transcription process of the gene, resulting in the inhibition of the function of the membrane protein SLC22A18, thus causing the occurrence of "allergy-short stature-fatty liver" imprinted gene syndrome

Method used

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  • Detection method of targeting membrane protein methylation as imprinting gene syndrome marker
  • Detection method of targeting membrane protein methylation as imprinting gene syndrome marker
  • Detection method of targeting membrane protein methylation as imprinting gene syndrome marker

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Embodiment 1

[0033] This embodiment discloses a detection method for targeting membrane protein methylation as an imprinted gene syndrome marker, including the following steps:

[0034] Step 1. Extract the cell-free DNA from whole blood. After passing the test, it is treated with bisulfite and used as an amplification template; the bisulfite treatment changes the unmethylated C in the DNA sample into U, and the methylated The C remains the same.

[0035] Step 2: Design and synthesize tag primers targeting membrane protein genes; the tag primers include a 5'-end modification group with a molecular tag, a nucleotide fragment for matching the amplification template, and a 3'-end modification group.

[0036] Step 3: Mix the amplification template and index primers for PCR amplification, connect to the special index sequencing adapters of the illumina sequencing platform, and construct amplicon libraries with different labels; the PCR amplification reagents include high-fidelity U-base-resistan...

Embodiment 2

[0046] Three comparison groups were selected, sample collection, synthesizing tag primers, building amplicon libraries, and on-machine sequencing were performed according to the detection method of Example 1, and then analyzed by bioinformatics to obtain the box plots and bee plots of the three comparison groups. group graphs (such as Figure 1-6 ) shown.

[0047] Differential analysis was carried out between each comparison group according to the test results, and the test results were consistent with clinical information.

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Abstract

The invention relates to a method for detecting targeted membrane protein methylation as an imprinting gene syndrome marker, which comprises the following steps: extracting whole blood free DNA (deoxyribonucleic acid), treating the whole blood free DNA by bisulfite after the whole blood free DNA is detected to be qualified, and using the whole blood free DNA as an amplification template; designing and synthesizing a tag primer of a targeted membrane protein gene; the method comprises the following steps: mixing an amplification template with a tag primer, carrying out PCR amplification, connecting an index sequencing joint special for an illumina sequencing platform, and constructing amplicon libraries of different tags; quantificationally mixing the amplicon libraries with different tags, and sequencing on a machine to obtain a sequencing result; the obtained sequencing result is analyzed through bioinformatics, and the methylation mutation condition in the target area is obtained; and detecting the site difference of each amplicon by adopting a t test method, counting the average methylation level of each amplicon, and carrying out differential analysis on each contrast group. The methylation sites of the imprinting gene SLC22A18 are screened by adopting an amplicon methylation sequencing method, and the method has the advantages of high accuracy, high flux, short period and low cost.

Description

technical field [0001] The invention belongs to the technical field of medical detection, and in particular relates to a detection method for targeting membrane protein methylation as an imprinted gene syndrome marker. Background technique [0002] Imprinted genes refer to the expression of homologous genes from only one parent, while the homologous genes from the other parent are not expressed. The existence of imprinted genes reflects the competition of sex. From the imprinted genes found so far, the contribution of the father to the embryo is to accelerate its development, while the mother is to limit the speed of embryo development. It is gender-specific to ensure the genetic advantage of its own genes. [0003] Aberrant expression of imprinted genes triggers a variety of human diseases with complex mutations and phenotypic defects. Studies have found that many imprinted genes have important regulatory effects on the growth and development of embryos and fetuses after ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858
CPCC12Q1/6883C12Q1/6858C12Q2600/154C12Q2523/125C12Q2535/122C12Q2531/113C12Q2537/165
Inventor 乔荆沈瑶殷俊
Owner SHANGHAI EAST HOSPITAL EAST HOSPITAL TONGJI UNIV SCHOOL OF MEDICINE
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