Application of methylation site, nucleic acid composition for detecting methylation, kit and detection method thereof
A technology of nucleic acid composition and detection reagent, which is applied in the field of molecular biology and can solve the problems of high false positive rate and unfavorable accurate detection of lung cancer
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Embodiment 1
[0093] Extract the DNA of CTC cells in the sample to be tested
[0094] (1) CTC cells were screened from the sample to be tested by a Hylo-Dva280 microfluidic chip, and the sample to be tested was whole blood.
[0095] (2) Using a commercial magnetic bead method DNA extraction kit (purchased from Sangon Bioengineering (Shanghai) Co., Ltd.) and following the instructions of the kit to extract DNA from CTC cells, the specific operations are as follows:
[0096] (a) Put the collected CTC cells into a sample tube, dilute to 50 μL with a buffer solution with salt balance function, add 430 μL of cell lysate, resuspend by shaking, and place in a water bath at 65°C for 5 minutes. After cooling to room temperature, 20 μL of RNaseA was added, mixed by a vortex shaker and left for 2 min. Add 400 μL of magnetic bead diluent and 10 μL of magnetic bead mother solution to the sample tube, shake and mix for 20 s with a vortex oscillator, and then let stand for 1 min.
[0097] (b) Place the ...
Embodiment 2
[0101] DNA methylation treatment of CTC cells in the sample to be tested
[0102] (1) Prepare a sodium bisulfite aqueous solution with a concentration of 3.6 mol / L, and titrate to pH 5.0 with a 3 mol / L NaOH aqueous solution.
[0103] (2) Take 25 μL of DNA to be treated, add 2.5 μL of 3mol / L NaOH aqueous solution, bathe in water at 42°C for 30 minutes, then add 15 μL of hydroquinone, keep in water bath until the solution turns light yellow. The above aqueous sodium bisulfite solution was added to bring the total solution volume to 300 μL. Keep the EP tube away from light, slowly invert and mix well, and then add 150 μL of petroleum jelly. Wrap it in tin foil to avoid light, and bathe in water at 50°C for 16 hours to obtain the DNA after methylation treatment, that is, the DNA to be tested.
Embodiment 3
[0105] Kits for detecting methylation
[0106]The kit includes nucleic acid composition, positive quality control, negative quality control, blank quality control, DNA polymerase, PCR reaction solution;
[0107] The nucleic acid composition is shown in Table 1. The nucleic acid composition includes a pair of amplification primers, a methylation detection probe and a non-methylation detection probe. Site area design, the methylation detection probe is designed for the methylated target gene at the 19730212 site on chromosome 7, and the unmethylation detection probe is designed for the 19730212 site on the 7th chromosome without methylation The target gene design of kylation, the target gene is CtDNA, the sequence of the 19730022-19730301 site region on chromosome 7 in the target gene is shown in SEQ ID No.5, specifically, the sequence shown in SEQ ID No.5 为:5'-GTCATGGTGGACGGATCACAAGGTCAGGAGATCGAGACCATCCTGGCTAACACGGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGTGGTGGCAGGCGCCTATGATC...
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