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Probe set for detecting tuberous sclerosis gene mutation and kit thereof

A probe set and kit technology, applied in the field of molecular biology, can solve problems such as heavy workload, high price, and low frequency of gene mutation

Active Publication Date: 2021-09-14
AEGICARE (SHENZHEN) TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, multiple ligation-dependent probe amplification (MLPA) technology is widely used in the market for analysis of large fragment abnormalities, which has high sensitivity, but it can only detect known disease-causing gene mutations, and MLPA also has its limitations. When the mutation or polymorphic region is close to the probe connection region or connection site, it will prevent the hybridization and connection of the probe, resulting in false positive results, and each known disease-causing region needs to design and synthesize specific probes, A lot of work and expensive
[0006] There are also genetic testing companies that use WES probes to capture the TSC1 / 2 gene and build a library for sequencing. Due to the lack of specific capture of the TSC1 / 2 gene, the sequencing depth of the TSC1 / 2 gene is greatly reduced. If the patient's TSC1 / 2 gene mutation is If it is chimeric, the frequency of gene mutation will be very low. In many cases, this low-frequency mutation will be treated as a false positive result. Although there are companies in the market that use ddPCR to detect low-frequency chimeric mutations, this method can only Detect known disease-causing mutations, and design primers for PCR for each mutation, which requires a lot of work

Method used

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  • Probe set for detecting tuberous sclerosis gene mutation and kit thereof
  • Probe set for detecting tuberous sclerosis gene mutation and kit thereof
  • Probe set for detecting tuberous sclerosis gene mutation and kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Such as figure 1 Shown is the detection flow chart of this embodiment, and the specific experimental process is as follows:

[0042] 1. Probe design and dilution

[0043] According to the liquid-phase hybridization capture technology, a set of DNA capture probes was designed. The length of the TSC1 gene is 53359bp, and a total of 394 probes were designed, and the length of each probe was about 110nt to 120nt, and the length of most probes was 120nt; It is 110nt~120nt, most of the probes are 120nt in length, and the probes cover all exons and exon-intron junction regions.

[0044] Dilute the dry probe powder synthesized by Integrated DNA Technologies with IDTE Buffer to a working solution with a concentration of 100 amol / μL. The specific dilution method is:

[0045] After centrifuging the dry powder of the probe, open the cap and add 100 μL IDTE (diluent prepared by Integrated DNA Technologies), mix well, and let it stand overnight at 4°C. At this time, the storage st...

Embodiment 2

[0093] Previously positive sample A1 (peripheral blood) provided by Shenzhen Anji Kanger Medical Testing Laboratory. The subject of this sample had recurrent bouts of stupor for 6 years, focusing on the investigation of tuberous sclerosis genes. It was adopted by Anji Kanger Medical Testing Laboratory Nanodigmbio's The detection result of the whole exome detection technology done by the Hybrid Capture Re agents kit is a TSC1 gene mutation, and the mutation site is NM_000368.4:c.1757del, which is detected by the method of this embodiment, such as figure 1 The detection flow chart is shown, and the specific detection method is as follows:

[0094] 1 DNA sample fragmentation

[0095] 1.1 Adoption dsDNA HS Assay Kit Fluorescent Quantitative Kit, check the sample concentration according to the operation of the quantitative kit, take 1 μg of qualified gDNA sample, and dilute the sample with 1×TE buffer solution (10mM Tris-HCl, 1mM EDTA, PH=8.0) to the specification volume of th...

Embodiment 3

[0260] Previously positive sample A2 provided by Shenzhen Anji Kanger Medical Laboratory. The subject of this sample was admitted to the hospital due to repeated convulsions for more than 5 years and fever for 1 day. Physical examination: a brown rash of about 3cm in size can be seen on the neck, showing a shark skin rash. There are multiple depigmentation spots all over the body. The initial diagnosis was tuberous sclerosis. The Shenzhen Anji Kanger Medical Testing Laboratory adopted Nanodigmbio's The detection result of the whole exome detection technology made by the Hybrid Capture Reagents kit was negative, and the method of Example 1 was used for detection. From the detection results, it was found that the TSC2 gene of sample A2 had a mutation in the intron region, and the mutation site It is NM_000548.3:c.848+281C>T(intron_variant), Intron9 / 40. Sanger sequencing was used to verify this site in the family sample, and the sequencing result was consistent with the detecti...

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Abstract

The invention relates to a probe set for detecting tuberous sclerosis gene mutation and a kit thereof. The probe set can specifically capture at least one of a TSC1 gene segment and a TSC2 gene segment. The probe set has the advantages of high coverage, high capture efficiency, high sequencing depth, high detection sensitivity and low detection cost, and can detect possible pathogenic new point mutation, copy number variation and chimeric mutation in a certain proportion.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a probe set and kit for detecting tuberous sclerosis gene mutation. Background technique [0002] Tuberous sclerosis complex (TSC) is an autosomal dominant genetic disease with an incidence rate of 1 / 6000-1 / 10000 in newborns and 1 / 8000 in adults. Tuberous sclerosis can lead to uncontrolled cell proliferation and differentiation, involving almost all organs and systems, especially the brain, skin, kidney, and heart. The pathological change is hamartoma. TSC1 or TSC2 are two important genes that cause tuberous sclerosis. TSC1 is located on chromosome 9 q34, contains 23 exons, and encodes hamartin, which consists of 1164 amino acids. TSC2 is located on chromosome 16 p13.3 and encodes a tuberin protein consisting of 1807 amino acids. Hamartin protein and Tuberin protein form a heterodimer (heterodimer) on the Golgi apparatus, that is, the Tuberin-Hamartin complex and function toge...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11C40B50/06
CPCC12Q1/6883C40B50/06C12Q2600/156Y02A50/30
Inventor 陈爽李妍珂刘永初刘超吕佩涛刘阳李阳
Owner AEGICARE (SHENZHEN) TECH CO LTD
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