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Library construction method for detecting endometrial cancer related gene mutation based on high-throughput sequencing

An endometrial cancer and library construction technology is applied in the field of library construction based on high-throughput sequencing to detect endometrial cancer-related gene mutations, and can solve the problems of heavy operator workload, large sample size requirements, and high sequencing costs. To achieve the effect of simple and convenient operation and reduce the detection cost

Active Publication Date: 2020-12-11
XIAMEN SPACEGEN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the commonly used method for detecting endometrial cancer-related genes MSH2, PMS2, MLH1, MSH6, EPCAM, TP53, POLE, and PTEN is to use Sanger sequencing method to design primers for these genes to measure gene sequences in separate tubes and segments for analysis. Whether there is a mutation, because the gene sequences of these 8 genes are relatively long, at least 200 amplification tubes are required for separate amplification and sequencing when the Sanger sequencing method is used for detection, which leads to a large demand for samples and a large workload for operators , long time to complete detection and high cost of sequencing per sample

Method used

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  • Library construction method for detecting endometrial cancer related gene mutation based on high-throughput sequencing
  • Library construction method for detecting endometrial cancer related gene mutation based on high-throughput sequencing
  • Library construction method for detecting endometrial cancer related gene mutation based on high-throughput sequencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] In this case, 100 cases of clinical endometrial cancer samples were collected, and the traditional Sanger sequencing method and the method of the present invention were carried out for double-blind comparison at the same time. In this example, 100 cases of clinical endometrial cancer samples were donated by the hospital. They were pathologically diagnosed as endometrial cancer samples. At the same time, family heredity was considered. After the samples were embedded in paraffin, 7 microns of 5 microns were excised. A white tablet was used as a comparison sample.

[0075] Utilizing the above method includes the following steps:

[0076] (1) Sample processing and template extraction quality control

[0077] Cut the wax block sample into 5-8 μm slices, and take 5 slices, or take 5 slices of the 5-8 μm slices that have been made. After dewaxing with xylene, use the paraffin-embedded DNA extraction kit of Meiji Company to extract genomic DNA. Steps according to the kit ins...

Embodiment 2

[0102] This example is to investigate the sensitivity and specificity of the method of the present invention, and the known common mutation sites in the following Table 6 are selected as the sequences for positive plasmid construction.

[0103] Table 6 Positive plasmids

[0104] serial number gene name Mutation COSMIC No. base change mutation type M1 TP53 COSM44957 c.80del deletion mutation M2 POLE COSM937332 c.857C>G point mutation M3 PTEN COSM1684696 c.38_39insC insertion mutation M4 EPCAM COSM4681226 c.924G>A point mutation M5 MSH6 COSM190062 c.1082G>A point mutation M6 MLH1 COSM6943688 c.105_107del deletion mutation M7 PMS2 COSM5621554 c.209A>G point mutation M8 MSH2 COSM5751788 c.89_90insT insertion mutation

[0105] The above mutant sequence plasmids were synthesized by genetic engineering technology, and the lengths were all 500bp.

[0106] 1. Sample pro...

Embodiment 3

[0112] In this example, the repeatability of the method of the present invention is investigated through the detection of clinical samples.

[0113] 10 cases of clinical endometrial cancer samples were collected and tested by traditional Sanger sequencing method at the same time. The mutation results are shown in Table 8.

[0114] Table 8 Sanger sequencing results

[0115]

[0116]

[0117] Utilizing the above method includes the following steps:

[0118] 1. Sample processing and template extraction quality control: cut the clinically collected wax block samples into 5-8 μm slices, take 5 slices, or take 5 slices of 5-8 μm slices that have been made, after xylene dewaxing, use Genomic DNA was extracted with the Paraffin-embedded DNA Extraction Kit of Meiji Company, and the specific steps were according to the operation instructions of the kit. The extracted DNA was dissolved in Tris-HCl (10mmol / L, pH 8.0), the quality of the extraction was detected by a UV spectrophotome...

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Abstract

The invention discloses a library construction method for detecting endometrial cancer related gene mutation based on high-throughput sequencing, and belongs to the technical field of biology. The method can be used for detecting mutation types of endometrial cancer related genes MSH2, PMS2, MLH1, MSH6, EPCAM, TP53, POLE and PTEN in specimens including fresh pathological tissues excised by operation, formaldehyde-fixed paraffin-embedded case tissues, paraffin sections, whole blood, plasma, serum, pleural effusion and the like, and single-tube rapid library construction can be carried out on multiple target sequences.The construction of the library is quickly completed, and the whole library construction process only needs 3 hours; The manual operation time is only 30 minutes, the difficulty that the mutation of the whole exon of the somatic cell polygene is required to be detected on the basis of a small amount of clinical samples in endometrial cancer samples clinically at present canbe effectively solved by combining with a high-throughput sequencing platform, and the cost is low.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a library construction method for detecting endometrial cancer-related gene mutations based on high-throughput sequencing. Background technique [0002] Endometrial cancer is a group of epithelial malignant endometrial cancers that occur in the endometrium, and is more common in perimenopausal and postmenopausal women. Endometrial cancer is one of the most common endometrial cancers of the female reproductive system, with nearly 200,000 new cases each year, and is the third most common gynecological malignant endometrial cancer (after ovarian cancer and cervical cancer). [0003] Although most endometrial cancers do not run in families, about 3 percent of endometrial cancers are inherited. Of these, Lynch syndrome is the most common cause. People with Lynch syndrome are more likely to develop endometrial, colorectal, and other cancers than people without the disorder. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C40B40/06C12Q1/6858
CPCC40B50/06C40B40/06C12Q1/6858C12Q2531/113C12Q2535/122C12Q2537/143C12Q2547/101C12N15/1093C12Q1/6886C12Q2600/156C12Q1/6806C12N15/111C12N2320/10C12Q1/686
Inventor 陈志宏肖冰陈琰康灿昆
Owner XIAMEN SPACEGEN BIOTECH CO LTD
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