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Ovarian premature senility related gene whole exon amplification and detection method

An all-exon, premature ovarian failure technology, applied in biochemical equipment and methods, microbial measurement/testing, etc., can solve problems such as abnormal three-dimensional structure of GDF9 protein, affecting translation, etc.

Inactive Publication Date: 2019-12-13
ZHEJIANG UNIV
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Problems solved by technology

GDF9 is the earliest identified ovum-derived factor. In one study, two heterozygous mutations of the GDF9 gene, p.D57Y and p.R146C, were found in Chinese ovarian hyporesponse patients, among which p.R146C was found in several previous studies. It has been reported that it only exists in patients with low ovarian response, but not in normal control patients, which mainly leads to abnormal three-dimensional structure of GDF9 protein, thereby affecting its post-transcriptional translation

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  • Ovarian premature senility related gene whole exon amplification and detection method
  • Ovarian premature senility related gene whole exon amplification and detection method
  • Ovarian premature senility related gene whole exon amplification and detection method

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[0050] Below in conjunction with specific embodiment the present invention will be described in further detail

[0051] The whole exon amplification and detection method of premature ovarian failure-related genes provided by the present invention can specifically and synchronously amplify and measure all exons of nine genes including FMR1, FOXL2, FSHR, POF1B, INHA, NOBOX, GDF9, BMP15 and FIGLA . The electrophoresis analysis of the amplification product is used to determine whether the amplification is successful, and then the sequencing analysis of the amplification product is used to determine whether there is a suspected disease-causing mutation.

[0052] Step 1: Genomic DNA Extraction

[0053] Obtain 5 mL of venous blood within 4 hours of blood collection from the hospital blood collection center (considered fresh blood), anticoagulate with EDTA, extract genomic DNA with a QIAGEN kit, dissolve it in double distilled water and store it at low temperature for later use.

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Abstract

The invention relates to the technical field of biological gene detection, and aims to provide an ovarian premature senility related gene whole exon amplification and detection method. The method comprises the following steps: extracting genome DNA from a detection material; using totally 120 primers shown in SEQ ID NO:1-120, carrying out an amplification test on all exons of 9 genes, namely FMR1,FOXL2, FSHR, POF1B, INHA, NOBOX, GDF9, BMP15 and FIGLA, wherein totally 61 PCR reactions in the amplification test and the negative control are carried out synchronously, and the Tm value range of the 120 primers is 57-62 DEG C; detecting an amplification product by adopting agarose electrophoresis and a gel imaging system; and analyzing the sequencing result of the amplification product by usingan automatic sequencer. All exons of 9 genes related to premature ovarian failure are synchronously amplified through optimized PCR amplification specificity, and a detection raw material is providedfor downstream detection. The method is simple and convenient to operate, short in amplification time, high in specificity and good in repeatability. The detected positive rate is greatly improved, and the application prospect is wide.

Description

technical field [0001] The invention relates to the technical field of biological gene detection, in particular to a method for amplifying and detecting the whole exon of a gene related to premature ovarian failure. Background technique [0002] Premature ovarian failure (POF) refers to women with normal or delayed menarche age and normal development of secondary sexual characteristics. Symptoms such as perimenopausal syndrome appear before the age of 40, amenorrhea lasts for more than 6 months, and reproductive organ atrophy , reduced sexual function or even infertility, increased mass concentrations of follicle stimulating hormone (FSH) and luteinizing hormone (LH), estradiol (Estradiol 2, E 2 ) is a gynecological endocrine disease with reduced mass concentration. [0003] Research on premature ovarian failure at home and abroad shows that patients with premature ovarian failure have some gene mutations at the genome level, and the research results on the relationship bet...

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6806
CPCC12Q1/6806C12Q1/6883C12Q2600/156C12Q2531/113
Inventor 张丹黄荷凤张润驹李静怡刘益枫陈松长徐谷峰
Owner ZHEJIANG UNIV
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