Primer and kit for detecting mutation and fusion of ret gene in thyroid cancer
A technology of thyroid cancer and primer pairs, which is applied in the determination/testing of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., and can solve the problems of simultaneous detection of RET gene mutation and fusion in thyroid cancer
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Embodiment 1
[0037] This embodiment relates to a primer combination that can be used to detect RET gene mutation and fusion of thyroid cancer, which is designed as follows:
[0038] (1) Primer design for RET gene mutation detection: According to the RET gene sequence information in the NCBI database, select the entire exon sequence of RET, and use Primer6.0 software to design multiple PCR primers required for amplification according to the basic principles of primer design;
[0039] (2) Primer design for RET gene fusion detection: Find the cDNA sequence information of the target genes CCDC6 and NCOA4 fused with the RET gene in thyroid cancer, and determine the DNA region where the CCDC6 gene and the RET gene are cleaved and fused, as well as the NCOA4 gene and RET gene. For the DNA region where the gene breaks and fuses, the primer combination is designed according to the relative positional relationship of the gene;
[0040] (3) Primer synthesis: synthesize the designed primers.
[0041]...
Embodiment 2
[0043] In this example, the primer combinations described in Table 2 above were used to establish an amplification library for sequencing. The specific library construction process is as follows ( figure 2 ):
[0044] (1) Sample extraction: extract DNA and RNA from patient samples, select the AllPrep DNA / RNA Mini Kit (QIAGEN) extraction kit to operate, and finally obtain DNA and RNA according to the kit extraction instructions, determine the concentration of the sample, and combine the DNA sample and RNA samples were diluted to 20ng / uL;
[0045] (2) RNA reverse transcription: select SuperScript III Reverse Transcriptase (Thermo Fisher) and RNaseOUT™ Recombinant Ribonuclease Inhibitor (Thermo Fisher) kits, prepare the reverse transcription reaction system according to Table 3, and set the reverse transcription reaction program according to Table 4. After the first step of reverse transcription was incubated at 65°C for 5 minutes, it should be placed on ice for 2 minutes. Afte...
Embodiment 3
[0066] In this example, the commercially available RET gene mutation DNA standard and RET gene fusion RNA standard were used to verify the performance of the finally optimized primer combination and library construction process of the present invention. At the same time, 30 clinical samples were collected, and commercially available kit products Aihuijian and Weihuijian were used to detect RET gene mutation and RET gene fusion respectively, and compared with the detection results of the present invention.
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