Probe set for detecting breakage of TERT (Telomerase Reverse Transcriptase) gene, kit and application of probe set

A gene fragmentation and probe set technology, which is applied in recombinant DNA technology, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of unsuitable for rapid detection of TERT genes in hospitals, complex sample processing, and long detection cycle. Achieve the effect of helping individualized treatment, high signal intensity, and high detection sensitivity

Active Publication Date: 2017-09-19
BEIJING CHILDRENS HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, at present, the detection of TERT fragmentation can only be based on whole genome sequencing technology, which not only requires complex sample processing, high professional requirements, long detection cycle and high cost, it is not suitable for hospitals to carry out rapid detection of TERT gene

Method used

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  • Probe set for detecting breakage of TERT (Telomerase Reverse Transcriptase) gene, kit and application of probe set
  • Probe set for detecting breakage of TERT (Telomerase Reverse Transcriptase) gene, kit and application of probe set
  • Probe set for detecting breakage of TERT (Telomerase Reverse Transcriptase) gene, kit and application of probe set

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Screening probe sets for detection of TERT gene disruption:

[0045] (1) Search for bacterial artificial chromosomes (BAC clones) corresponding to both sides of the TERT gene on chromosome 5 (ie, the telomere side and the centromere side) through http: / / genome.ucsc.edu / . The results showed that there are dozens of BAC clones covering the 500 KB at both ends of the TERT gene (chr5:1,253,287-1,295,162), but these clones have not been verified and lack specific descriptions.

[0046] (2) Probe design principle: Select multiple BAC clones of similar size on both sides of the gene breakpoint, control the farthest distance between the BAC clone fragments on both sides within 1500Kb and do not overlap each other, and multiple fragments on the same side interact with each other. There is a certain sequence overlap between them to avoid signal dispersion caused by large gaps. According to the above probe design principles, it is planned to select three BAC clones on one side of...

Embodiment 2

[0055] Preparation of fluorescently labeled BAC clone probe set for detection of TERT gene disruption:

[0056] The corresponding BAC clones were purchased from Invitrogen, and the plasmids were extracted after the BAC clones were cultured. Using the gap translation method, the two BAC clone fragments RP11-1107M2 and RP11-260H6 on the telomere side were labeled with green fluorescence with Sptectrum green-dUTP. Two BAC clone fragments RP11-325I22 and RP11-768M3 on the centromere side were labeled with red fluorescence by Sptectrumorange-dUTP. Prepare the labeled probe, Human cot-1 DNA, hybridization buffer, and purified water in proportion to make a probe hybridization solution, and store it in a dark freezer at -20°C.

Embodiment 3

[0058] Prepare a kit for detecting TERT gene disruption:

[0059] The probe hybridization solution prepared in Example 1 and the DAPI dye used for nuclear DNA staining constitute a kit.

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Abstract

The invention provides a probe set for detecting the breakage of a TERT (Telomerase Reverse Transcriptase) gene, a kit and application of the probe set to preparation a reagent or the kit for identifying a high-risk patient suffered from neuroblastoma. The probe set provided by the invention comprises clonal fragments RP11-325I22, RP11-768M3, RP11-1107M2 and RP11-260H6, wherein the RP11-325I22 and the RP11-768M3 are marked with fluorescent dyes of the same color; the RP11-1107M2 and the RP11-260H6 are marked with the fluorescent dyes of another color. The breakage of the TERT gene can be quickly, accurately and sensitively detected.

Description

technical field [0001] The invention relates to the field of in situ hybridization, in particular to a probe group, a kit and an application thereof for detecting TERT gene breaks. Background technique [0002] Neuroblastoma (NB) belongs to peripheral sympathetic nervous system tumors in children and is the most common embryonal extracranial tumor in children. NB tumors have high malignancy and rapid progression, and are prone to metastasis to bone marrow, bone and other organs, accounting for up to 15% of childhood tumor-related deaths. Even after multiple treatments such as chemotherapy, surgery and radiotherapy, NB tumors still recur in about 60% of cases. About 40% of children with Shenmue tumors are diagnosed as high-risk NB, and their 5-year survival rate is only 30%-50%. Due to the lack of effective treatment, poor prognosis, high recurrence rate and mortality, NB brings heavy economic losses and burdens to children's families and society. Therefore, in-depth resea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q2600/166
Inventor 于永波倪鑫郭永丽何乐建王焕民金雅琼杨业然陈绍宇
Owner BEIJING CHILDRENS HOSPITAL AFFILIATED TO CAPITAL MEDICAL UNIV
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