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Sexual dysplasia-related gene capture kit and application thereof

A gene capture and kit technology, applied in the fields of biochemical equipment and methods, DNA/RNA fragments, and microbial determination/examination, which can solve the problems of large differences in clinical phenotypes and difficult diagnosis.

Active Publication Date: 2020-03-24
ZHEJIANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Different etiologies may have the same or similar clinical manifestations, and the clinical phenotypes caused by the same etiology at different times or different degrees of influence are very different, which brings certain difficulties to the diagnosis

Method used

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  • Sexual dysplasia-related gene capture kit and application thereof
  • Sexual dysplasia-related gene capture kit and application thereof
  • Sexual dysplasia-related gene capture kit and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1. Preparation of a complete set of reagents and kits for detecting sex chromosome abnormality-related genes

[0099] 1. Design and preparation of sex chromosome abnormality-related gene capture probes

[0100] 1. Design and synthesize 277 probes based on the whole exon sequences of 277 sex chromosome abnormality-related genes (design 120bp probe sequences for non-repeated regions in each region, and move each sequence along the gene position to design) .

[0101] Each of the 277 probes is composed of DNA fragment 1, probe fragment, and DNA fragment 2 (each probe is DNA fragment 1, probe fragment and DNA fragment 2 in turn from the 5' end to the 3' end, 277 DNA Fragment 1 among the probes is the same, and DNA Fragment 2 is the same among the 277 probes). The nucleotide sequence of DNA fragment 1 is 5'-GACTACATGGGACAT-3'. The nucleotide sequence of DNA fragment 2 is 5'-GGAACCTACGACGTA-3'. The probe fragments in the 277 probes are shown in Table 1.

[0102] T...

Embodiment 2

[0129] Example 2. Establishment of the method of using the kit for capturing genes related to abnormal sexual development

[0130] 1. Sample library preparation

[0131] 1. With the informed consent of the patient, extract the genomic DNA from the peripheral blood of the patient to be tested.

[0132] 2. Fragment the genomic DNA obtained in step 1 to 200-400bp fragments, and use the NGS Fast DNALibrary Prep Set for Illumina kit (Kangwei reagent, catalog number: CW2585M) to construct a library to obtain a DNA gene library.

[0133] 2. Sample enrichment hybridization

[0134] 1. Preparation of hybridization solution

[0135] Hybridization solution (100μl): 250ng DNA library prepared in step 1, 10μl enrichment buffer BL, 5μl biotin-labeled probe set obtained in step 1 (probe concentration is 100ng / μL), 37μL preheated enrichment Buffer solution HY (preheated at 65°C, shake well to resuspend the pellet before use).

[0136] 2. Hybridization

[0137] Take the hybridization solu...

Embodiment 3

[0168] Example 3, Sequencing Effect Verification

[0169] With the informed consent of the patients, peripheral blood samples from 2 patients with genes related to chromosomal abnormalities were collected and tested according to the method in Example 2.

[0170] The results are shown in Table 2. The results confirmed that more than 99% of the original short sequences can be compared back to the reference sequence of the target region, the capture efficiency is above 35%, and the average sequencing depth of the target region is greater than 250X, fully meeting the requirements of general genetic disease diagnosis.

[0171] Table 2 Data Analysis Results

[0172]

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PUM

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Abstract

The invention discloses a sexual dysplasia-related gene capture kit and an application thereof. The sexual dysplasia-related gene capture kit carries out target region sequencing analysis on the wholeexon group or the sexual dysplasia-related genome (respectively accounting for 1% and 0.01% of the whole genome) of a patient with hereditary dysplasia, most pathogenic mutation information of the disease is captured, and the kit has the advantages of small required sample amount, low cost and high throughput, can detect many samples, and promotes discovery of new pathogenic variation of hereditary dysplasia. The sexual dysplasia-related gene mutation site selected by the invention covers common hot spot mutation and other unusual mutation sites of Chinese people. The high throughput of the kit makes sexual dysplasia-related genes in people comprehensively screened, so the kit is of great significance to improving population quality and benefiting the society.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a gene capture kit related to abnormal sexual development and its application. Background technique [0002] Sexual development and differentiation is a complex and continuous process, starting from the fertilized egg and ending with the maturity and perfection of secondary sexual characteristics at puberty, including sex determination, differentiation and development of gonads, internal reproductive tract and external genitalia. The normal development and differentiation of gonads is determined by the following three factors: ①Sex chromosomes (xY, XX), which determine the genetic sex of an individual, are the innate basis of gonad differentiation and development; ②Related factors that regulate the formation and differentiation of sexual organs in utero, Genetic mutations of these factors lead to dysfunction of the expressed proteins, which can directly affect intrauterine sexual dev...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6806C12Q1/6869C12N15/11
CPCC12Q1/6806C12Q1/6869C12Q1/6883C12Q2600/156C12Q2565/519C12Q2531/113C12Q2535/122
Inventor 傅君芬伍建姬晓雯江凯王海丽
Owner ZHEJIANG UNIV
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