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DNA (Deoxyribonucleic Acid) methylation marker for evaluating early abortion risk, primer and application thereof

A methylation marker, early abortion technology, applied in the field of genetic engineering, to achieve the effect of improving sensitivity and specificity, quantitatively accurate, fast and accurate grasp of disease state and disease severity

Active Publication Date: 2018-07-24
NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the application of blood DNA methylation in the auxiliary diagnosis of early miscarriage. If the miscarriage-related DNA methylation can be screened as a biomarker, it can quickly assess the risk of early miscarriage and provide a basis for prevention and treatment

Method used

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  • DNA (Deoxyribonucleic Acid) methylation marker for evaluating early abortion risk, primer and application thereof
  • DNA (Deoxyribonucleic Acid) methylation marker for evaluating early abortion risk, primer and application thereof
  • DNA (Deoxyribonucleic Acid) methylation marker for evaluating early abortion risk, primer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 Research object selection and sample collection

[0041] (1) According to the diagnostic criteria: pregnant women with pregnancy loss before 20 weeks of gestation were the case group; according to age, gestational weeks, BMI and other indicators, pregnant women with normal fertility and non-medical reasons for abortion were selected by frequency matching method (no abortion) Signs and signs, no history of miscarriage, and at least one normal child was born) as the control group.

[0042] (2) B-ultrasound shows no germ or fetal heartbeat, and abortion caused by factors such as monogenic genetic diseases, polygenic genetic diseases, chromosomal abnormalities, and gene mutations must be excluded.

[0043] (3) Grouping of research objects:

[0044] Group A: the control group of pregnant women with abortion for non-medical reasons (n=44, 4 people were sequenced and screened, 20 people carried out independent verification of tissue DNA methylation, and 20 people...

Embodiment 2

[0048] Example 2 Differential Analysis of DNA Methylation in Villi Tissue

[0049]QiagenDNeasy Blood&Tissue Kit to extract genomic DNA, the steps are as follows: Grind the villous tissue with a grinder, resuspend in 200 μL PBS, add 20 μL proteinase K and 200 μL Buffer AL, shake and mix, and incubate at 56 °C for 10 minutes; add 200 μL ethanol, Shake and mix well; transfer the mixture to a DNeasy Mini filter column in a 2mL centrifuge tube, centrifuge at 6000g for 1 minute, discard the filtrate and the centrifuge tube; put the filter column into a new 2mL collection tube, add 500μL BufferAW1, Centrifuge at 6000g for 1 minute, discard the filtrate and collection tube; put the filter column into a new 2mL collection tube, add 500μL Buffer AW2, centrifuge at 20000g for 3 minutes, discard the filtrate and collection tube; move the filter column to a new 1.5mL or 2mL centrifuge tube; add 100μL Buffer AE to the center of the filter column membrane to elute DNA, incubate at room tempe...

Embodiment 3

[0057] Example 3 Verification of Differential DMR in Independent Population

[0058] Select 20 independent population controls and 20 abortion cases, and use the QiagenDNeasy Blood&Tissue kit to extract genomic DNA. The steps are as follows: Grind the villous tissue with a grinder, resuspend it with 200 μL PBS, add 20 μL proteinase K and 200 μL Buffer AL, shake and mix well , incubate at 56°C for 10 minutes; add 200 μL of ethanol, shake and mix; transfer the mixture to a DNeasy Mini filter column in a 2mL centrifuge tube, centrifuge at 6000g for 1 minute, discard the filtrate and centrifuge tube; put the filter column in into a new 2mL collection tube, add 500μL Buffer AW1, centrifuge at 6000g for 1 minute, discard the filtrate and the collection tube; put the filter column into a new 2mL collection tube, add 500μL Buffer AW2, centrifuge at 20000g for 3 minutes, discard Remove the filtrate and collection tube; transfer the filter column to a new 1.5mL or 2mL centrifuge tube; a...

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Abstract

The invention belongs to the field of the genetic engineering, and particularly provides a blood DNA (Deoxyribonucleic Acid) methylation marker for evaluating an early abortion risk, a primer and theapplication thereof. The DNA methylation marker comprises eight DNA methylation sites positioned near a PRDM1 gene transcription starting area: cg25608130, cg19577529, cg10573915, cg11072009, cg02667677, cg23778363, cg24793124 and cg07331652. The successful development of the category of the differentiated DNA methylation biological marker is the overturn of a traditional biomarker which gives a priority to protein, a brand new situation is started for diagnosing, preventing and curing the early abortion, and a reference is provided for researching the biomarkers of other diseases. The DNA methylation marker has the advantages of abundant raw materials and convenience in operation and is the biomarker with prospect.

Description

field of invention [0001] The invention belongs to the field of genetic engineering, and specifically relates to a group of DNA methylation markers, primers and applications thereof for assessing the risk of early miscarriage and assisting in the diagnosis of early miscarriage. Background technique [0002] Spontaneous abortion (Spontaneous Abortion / Pregnancy Loss) refers to the clinical pregnancy loss before the 20th week of pregnancy or the pregnancy loss of the fetus weighing less than 500g. It is a relatively common pathological pregnancy in clinical practice, with an incidence rate of about 10%-15%. As a pathological pregnancy, spontaneous abortion has brought great harm to the physiology, psychology and family of women of childbearing age. At present, the specific pathogenesis of early miscarriage is still unclear, and the treatment methods have little effect. Therefore, it is particularly important to study the specific pathogenesis of early miscarriage, so as to ma...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/154
Inventor 杜桂珍于明明黄振遥韩莉傅广波夏彦恺王心如
Owner NANJING MEDICAL UNIV
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