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Thyroid cancer pathogenicity-related gene fusion and mutation detection kit

A detection kit and a technology for thyroid cancer, which are applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of low expression abundance and high cost of whole transcriptome sequencing, and achieve wide and easy availability of raw materials, and solve Detecting costly effects

Active Publication Date: 2017-04-26
INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

However, in the human transcriptome, there are more than 20,000 genes. In order to detect all potential gene fusions, at least 8Gb of transcriptome sequencing data is required; if the expression abundance of gene fusions in the transcriptome is low , even more transcriptome sequencing data are needed
Therefore, in actual clinical diagnosis, facing the problem of high cost of whole transcriptome sequencing

Method used

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  • Thyroid cancer pathogenicity-related gene fusion and mutation detection kit
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  • Thyroid cancer pathogenicity-related gene fusion and mutation detection kit

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preparation example Construction

[0030] The present invention also provides a method for preparing probes in the RNA probe set, preferably comprising the following steps:

[0031] 1) For the coding sequence of the gene, from the 5' to the 3' direction, according to the principle of sequence reverse complementarity, design a probe sequence with a length of 110-130bp from the first base, and every two adjacent probe sequences There is a 50-70bp overlap between the needle sequences;

[0032] 2) At the 5' end and 3' end of each probe sequence, add a primer whose sequence is Seq ID No170 and a primer whose sequence is SeqID No171, respectively, to form a probe set sequence list with the same sequence at both ends;

[0033] 3) Using oligonucleotide in situ synthesis technology to perform large-scale synthesis of oligonucleotides on the chip to the sequences in the above-mentioned probe set sequence list;

[0034] 4) eluting the oligonucleotides on the chip to form an oligonucleotide mixture;

[0035] 5) using the...

Embodiment 1

[0119] Screening of genes related to pathogenicity of thyroid carcinoma

[0120] By investigating all the papers on the genetics of thyroid cancer on the medical literature retrieval service system PUBMED so far, five pathogenic genes related to thyroid cancer were identified, specifically RET, NTRK1, NTRK3, PPARγ, and RAF1.

[0121] Probe set design and preparation

[0122] According to the above 5 gene sequences (HG19 version), those skilled in the art can design and synthesize probe sequences by known and general methods. In this example, the synthesized probe sequences were amplified, in vitro transcribed and biotin-labeled. Specific steps are as follows:

[0123] 1. For the coding sequence of the gene, from the 5' to the 3' direction, according to the principle of sequence reverse complementarity, design a probe sequence with a length of 110-130bp from the first base, and every two adjacent probe sequences There is a 50-70bp overlap between needle sequences;

[0124] 2...

Embodiment 2

[0137] Use Trizol or similar products to extract RNA from tumor tissue. Quantify with Nanodrop, agarose gel electrophoresis, and check the quality of the sample. The complete genomic DNA electrophoresis band should usually not be less than 20kb, and the complete RNA electrophoresis band should be clear 28S, 18S and 5S bands.

[0138] 2 ug of qualified RNA was reverse-transcribed with NEB Protoscript II Reverse Transcriptase (purchased from NEB Company) to synthesize single-stranded cDNA. Double-stranded cDNA was synthesized with NEBNextmRNA Second Strand Synthesis Module (purchased from NEB Company). Double-stranded cDNA was purified with DNA Clean & Concentrator Kit (purchased from Zymo). The double-stranded cDNA was randomly broken into small fragments of 150-250bp using the ultrasonic breaker Bioruptor pico;

[0139] Pre-capture small fragment library preparation was performed using the Illumina TruSeq DNA Library Prep Kit.

[0140] Take 750ng of the small fragment libra...

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Abstract

Disclosed is a thyroid cancer pathogenicity-related gene fusion and mutation detection kit. The thyroid cancer pathogenicity-related gene fusion and mutation detection kit is characterized by comprising an RNA probe set, primers, blocking buffer, an RNA enzyme blocking solution, hybridization buffer, binding buffer, 1x SSC rinse liquid, 0.1x SSC rinse liquid, PCR reaction liquid and TE buffer, wherein the RNA probe set comprises 161 RNA probes with the sequences shown as Seq ID No.1-161. The thyroid cancer pathogenicity-related gene fusion and mutation detection kit is capable of detecting 5 disease-related pathogenic genes simultaneously, providing conditions for precise diagnosis and treatment of individuals suffering from thyroid cancer and evaluating cancer risks of thyroid nodule patients.

Description

technical field [0001] The invention relates to the field of gene variation detection, in particular to a detection kit for thyroid cancer pathogenicity-related gene fusion variation. Background technique [0002] Thyroid cancer is the most common malignant disease in the endocrine system, and its incidence has tripled in the past three decades. According to the 2012 statistics from the Registry of the National Cancer Center of China, the incidence of thyroid cancer in my country is about 8.76 per 100,000, and about 118,000 people are diagnosed with thyroid cancer every year. Thyroid cancers can be divided into the following subtypes based on cellular origin and clinicopathology: well-differentiated papillary and follicular carcinomas arising from thyroid follicular epithelium, poorly differentiated carcinomas, and undifferentiated carcinomas; and carcinomas originating in thyroid follicles. Medullary thyroid carcinoma of parafollicular cells. Papillary carcinoma accounts ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 梁加龙滕花景李津臣孙中生
Owner INST OF ZOOLOGY CHINESE ACAD OF SCI
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