Primer for detecting thyroid cancer pathopoiesia related gene variation on basis of high throughput sequencing technology and application of primer

A disease-related gene, thyroid cancer technology, applied in the field of molecular biology, can solve the problems of complex operation and high development cost, and achieve the effects of high sensitivity, simple operation and high accuracy

Pending Publication Date: 2019-02-22
HANGZHOU D A GENETIC ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Targeted capture technology is suitable for targeted capture of big data, but the development cost is high and the operation is complicated; multiplex PCR capture technology is easy to operate, flexible, and suitable for small and medium throughput data volumes

Method used

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  • Primer for detecting thyroid cancer pathopoiesia related gene variation on basis of high throughput sequencing technology and application of primer
  • Primer for detecting thyroid cancer pathopoiesia related gene variation on basis of high throughput sequencing technology and application of primer
  • Primer for detecting thyroid cancer pathopoiesia related gene variation on basis of high throughput sequencing technology and application of primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Amplify the nucleic acid sequence of the thyroid cancer-related gene mutation site, which was synthesized by Platinum Biotechnology (Shanghai) Co., Ltd.:

[0060] Primer pairs for amplifying BRAF:

[0061] BRAF_F1 (SEQ ID NO.1):

[0062] 5'-ACACGACGCTCTTCCGATTCTTTTACTTACTACACCTCAGA-3',

[0063] BRAF_R1 (SEQ ID NO. 2):

[0064] 5'-GACGTGTGCTCTTCCGATCTGATGGGACCCACTCCATCGAGA-3';

[0065] Primer pairs for amplifying KRAS:

[0066] KRAS_F1 (SEQ ID NO. 3):

[0067] 5'-ACACGACGCTCTTCCGATCTCTAATATAGTCACATTTTCATT-3',

[0068] KRAS_R1 (SEQ ID NO.4):

[0069] 5'-GACGTGTGCTCTTCCGATCTAGCTGTATCGTCAAGGCACTC-3';

[0070] KRAS_F2 (SEQ ID NO.5):

[0071] 5'-ACACGACGCTCTTCCGATCTTGGTCCCTCATTGCACTGTACTC-3',

[0072] KRAS_R2 (SEQ ID NO. 6):

[0073] 5′-GACGTGTGCTCTTCCGATCTTGTTTCTCCCCTTCTCAGGATTC-3′;

[0074] KRAS_F3 (SEQ ID NO. 7):

[0075] 5'-ACACGACGCTCTTCCGATCTAGATCTGTATTTATTTCAGTG-3',

[0076] KRAS_R3 (SEQ ID NO.8):

[0077] 5'-GACGTGTGCTCTTCCGATCTAACAGTAGACACAAA...

Embodiment 2

[0144] Embodiment 2: the preparation method of kit.

[0145] (1) High-fidelity PCR enzyme mixture: the reaction solution contains enzyme buffer, Mg 2+ , dNTPs, high-fidelity DNA polymerase, stored at -20°C;

[0146] (2) DNA-specific primer mixing pool: the reaction solution contains DNA amplification-specific primers, including SEQ ID NO.1-22, at a concentration of 10uM, and stored at -20°C;

[0147] (3) RNA-specific primer mixing pool: the reaction solution contains specific primers for cDNA amplification, including SEQ ID NO.25-36, the concentration is 10uM, and stored at -20°C;

[0148] (4) I5XX primer: the reaction solution contains a universal primer with i5XX index, the concentration is 10uM, and stored at -20°C;

[0149] (5) I7XX primer: the reaction solution contains a universal primer with i7XX index, the concentration is 10uM, and stored at -20°C;

[0150] (6) Negative control: RNase-free water.

Embodiment 3

[0151] Embodiment 3: detection method.

[0152] Instruments: qualitative PCR instrument, vortex mixer, 24-well high-speed centrifuge, Qubit fluorescence quantitative instrument, Agilent 4200 fragment analyzer, pure water instrument, second-generation high-throughput sequencer.

[0153] (1) Co-extraction of sample DNA and RNA: Use Qiagen AllPrep DNA / RNA FFPE Nucleic Acid Extraction Kit (Cat. No.: 80234) and refer to the kit instructions to extract DNA and RNA from the sample. Sample DNA is used as nucleic acid constant temperature amplification reaction The template is spare.

[0154] (2) Sample RNA is reverse transcribed to prepare cDNA: the extracted RNA sample uses TAKARA PrimeScript TM RTReagent Kit with gDNA Eraser (Perfect Real Time) (Product No.: RR047A), refer to the kit instructions for operation, reverse transcribe into cDNA, and cDNA is used as template for nucleic acid constant temperature amplification reaction.

[0155] (3) Use this kit for library construction...

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Abstract

The invention provides a kit for detecting thyroid cancer pathopoiesia related gene variation on the basis of a high throughput sequencing technology. The kit is used for variation detection of thyroid cancer related genes, including 18 mutation sites of 6 genes comprising BRAF, NRAS, KRAS, HRAS, TERT and TP53 and 3 fusion genes comprising CCDC6/RET, NCOA4/RET and PAX8/PPARg. Online sequencing libraries are constructed by two round of multiple PCR, the specific primer is designed for target areas of the nine genes to be used in the first round of multiple PCR, and a general sequencing sequenceprimer is used in the second round of multiple PCR. The invention also discloses a kit, including primer sequences and library systems, for detecting thyroid cancer pathopoiesia related gene variation, and the kit can assist in diagnosing equivocal thyroid sarcoidosis lesions.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a primer for building a library, a method for building a library, and a kit for detecting gene mutations related to thyroid cancer pathogenesis based on high-throughput sequencing technology. Background technique [0002] According to statistics, the incidence of thyroid nodules detected by palpation is 5%, and that by ultrasonography is 35%. 85% to 95% of these nodules are benign nodules, such as nodular goiter and thyroid adenoma, and only 5% to 15% of the nodules are malignant nodules, that is, thyroid cancer. In the diagnosis of thyroid cancer, fine-needle aspiration cytology (FNA) is the most reliable means to evaluate whether thyroid nodules are malignant tumors. However, FNA cannot definitively diagnose 20%-30% of thyroid nodules, and such cases have been reported as indeterminate. Due to the lack of reliable diagnostic methods, some patients need to confirm th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6806C12Q1/6869C12N15/11
CPCC12Q1/6806C12Q1/6869C12Q1/6886C12Q2600/156C12Q2600/16C12Q2531/113C12Q2537/143C12Q2535/122
Inventor 任绪义张锋赵铃铃金亚南
Owner HANGZHOU D A GENETIC ENG
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