Group of thyroid cancer markers and application thereof
A thyroid cancer and marker technology, applied in the direction of recombinant DNA technology, microbial measurement/testing, biochemical equipment and methods, etc., can solve the problems of inability to obtain molecular information, treatment options or prognosis evaluation without guiding significance
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Embodiment 1
[0157] Example 1 gDNA extraction
[0158] Most of the detection samples of patients with thyroid disease are biopsy puncture samples, the sample size is small, and the quality of nucleic acid extraction is very important. In this example, the papillary thyroid carcinoma sample (tissue and blood) numbered BT1809140101LNDBE / TV is taken as an example to conduct genome analysis. DNA (gDNA) extraction was carried out using QIAGEN’s tissue and blood DNA extraction kit, the extraction steps were carried out according to the instructions, and the extracted nucleic acids were quality controlled using Qubit 3.0.
[0159] The extracted nucleic acid was subjected to enzyme digestion and fragmentation. The enzyme digestion system was shown in Table 1, and the enzyme digestion conditions were shown in Table 2. After the enzyme digestion reaction was completed, 40 μL of enzyme-free water was added to the enzyme digestion product to a total volume of 50 μL. Agilent 2100 was used to check the ...
Embodiment 2
[0165] Embodiment 2 library construction
[0166] This embodiment builds a database with reference to the SureSelect XT Low Input System, and improves and optimizes the steps. The specific operations are as follows:
[0167] (1) End repair
[0168] Add enzyme-free water (Nuclease-free Water) to the fragmented sample obtained in Example 1 to make the volume up to 50 μL, prepare the end repair reaction system according to Table 3, mix it and place it on a PCR instrument at 20°C for 15 minutes, 72 Keep at ℃ for 15min, and store at 4℃ for later use.
[0169] Table 3 End repair reaction system
[0170] Reagent Dosage (μL) Buffer (End Repair-ATailing Buffer) 16 End Repair-ATailing Enzyme Mix 4 Fragmented samples 50 total capacity 70
[0171] (2) Joint connection
[0172] Prepare the adapter connection reaction system according to Table 2, mix it and place it on the PCR instrument for 30 minutes at 20°C, and store it at 4°C for later use; i...
Embodiment 3
[0192] Example 3 Target region capture of thyroid-related genes
[0193] This embodiment carries out target area capture with reference to SureSelect XT Low Input System, specific operation is as follows:
[0194] (1) capture hybridization
[0195] Transfer 1000ng of the pre-amplified library (including capture probes such as SEQ ID NO: 1 to 44) to a new centrifuge tube, dry it, and add 12 μL ddH 2 O dissolved, vortexed;
[0196] Add 5 μL of blocking solution (SureSelect XT HS and XT Low Input Blocker Mix), vortex to mix, transfer to a PCR plate, and place on a PCR machine for reaction, the conditions are shown in Table 9; when the program runs to step 3, pause , add 13 μL of the capture hybridization system shown in Table 10.
[0197] Table 9 target region capture reaction conditions
[0198]
[0199] Table 10 capture hybrid system
[0200] Reagent Dosage (μL) RNase Blocking Solution (25% RNase Block solution) 2 Library (Capture library 2 ...
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