Gene mutants and their applications

A gene and biological sample technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of recurrent miscarriage that need to be deepened

Active Publication Date: 2019-11-26
SHENZHEN HUADA GENE INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Therefore, the current research on recurrent miscarriage still needs to be in-depth

Method used

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  • Gene mutants and their applications
  • Gene mutants and their applications
  • Gene mutants and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0366] Example 1 Whole Exome Sequencing Determination of Disease-causing Genes and Mutation Sites

[0367] 1. Sample collection:

[0368] The inventor collected 96 cases of recurrent miscarriage, all of which were sporadic cases, and no abnormalities were found in age factors, living habits, immune factors, infection factors, endocrine factors, reproductive system factors, etc. in clinical testing, but all patients had It is manifested as 2 or more spontaneous abortions in a row.

[0369] 2. Whole exome sequencing to determine the pathogenic gene and mutation site

[0370] The inventors used the NimbleGen SeqCap EZ Human Exome Library v2.0 whole exome capture platform, combined with Comlete Genomics high-throughput sequencing technology, to perform whole exome capture sequencing on the above 96 cases of recurrent miscarriage. The specific steps are as follows:

[0371] 2.1 Sample preparation

[0372] The peripheral blood of the above-mentioned 96 cases of recurrent miscarri...

Embodiment 2

[0381] Example 2 Sanger method sequencing to verify the pathogenic mutation of recurrent miscarriage

[0382] Due to a certain degree of false positives in the sequencing of the target region, the inventors used the Sanger sequencing method to verify 4 heterozygous rare mutation sites that may have pathogenic significance.

[0383] The 4 new mutations obtained in the detection of Example 1 were detected respectively, and primers were designed for the sequences of the 4 new mutation sites involved in the ESR2 and AR genes, and then the above mutations were obtained by PCR amplification, product purification and sequencing. Regarding the sequence, it is determined whether the sequence determination results belong to the mutant or wild type, whether it is a heterozygous mutation or a homozygous mutation, and the correlation between the sequence and phenotype and recurrent miscarriage.

[0384] The specific method steps are as follows:

[0385] 1. DNA extraction

[0386] Accordi...

Embodiment 3

[0410] Embodiment 3 detection kit

[0411] Prepare a detection kit comprising primers suitable for detecting at least one of ESR2 and AR gene mutants (wherein, compared with SEQ ID NO: 1, has at least one selected from the following mutations: c.1501C>T mutation and c.17C>T mutation, or compared to SEQ ID NO: 2 has at least one mutation selected from the group consisting of: c.270G>T and c.1191C>G,) so as to be used for screening biological samples susceptible to recurrent miscarriage, wherein the specific sequences of these primers are shown in Example 2.

[0412] The specific steps for using the above kit to screen biological samples susceptible to recurrent miscarriage are: extract the DNA of the test subject according to the method described in step 1 of Example 2, use the extracted DNA as a template and the above-mentioned exon-specific primers Carrying out a PCR reaction, purifying the PCR product according to conventional methods in the art, sequencing the purified p...

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Abstract

The invention discloses a gene mutant and application thereof. Compared with SEQ ID No. 1, the gene mutant has at least one mutation selected from a group consisting of c.1501C>T mutation and c.17C>T mutation; or compared with SEQ ID No. 2, the gene mutant has at least one mutation selected from a group consisting of c.270G>T mutation and c.1191C>G mutation. Whether a biological sample is prone to recurrent abortion can be effectively detected by detecting existence of the gene mutant in the biological sample.

Description

technical field [0001] The present invention relates to gene mutants and applications thereof. In particular, the present invention relates to isolated nucleic acids, isolated polypeptides, methods of screening biological samples for susceptibility to recurrent miscarriage, systems for screening biological samples for susceptibility to recurrent miscarriage, biological samples for screening of susceptibility to recurrent miscarriage kits, constructs, and recombinant cells. Background technique [0002] Clinically, two or more consecutive spontaneous abortions of less than 20 weeks of pregnancy are defined as recurrent spontaneous abortions (RSA). RSA may be caused by many factors, such as age factors, living habits, genetic factors, immune factors, infection factors, endocrine factors, reproductive system factors and so on. In addition to the known factors, about 50% of the causes of RSA are still unknown, and this type of unexplained recurrent miscarriage that has exclude...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/72C12M1/00C12Q1/6883C12N15/63C12N5/10C12N1/21C12R1/19
CPCC07K14/721C12Q1/6883C12Q2600/156
Inventor 王文婧徐凤萍杨振军袁剑颖陈苑叶玲飞蒋慧
Owner SHENZHEN HUADA GENE INST
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