Gene mutation site group of NK/T cell lymphoma, targeted sequencing kit and application
A technology for targeted sequencing and lymphoma. It is applied in recombinant DNA technology, DNA/RNA fragments, and the determination/inspection of microorganisms. The effect of competence and efficiency
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Embodiment 1
[0062] The reagents used in this example are specifically shown in Table 3, and other reagents are commercially available reagents unless otherwise specified.
[0063] table 3
[0064]
[0065]
[0066] 1. Sample acquisition
[0067] 102 patients with NK / T cell lymphoma were willing to undergo molecular testing and obtained informed consent. Frozen tissue samples, paraffin tissue samples, or biopsy tissue samples were obtained from the patients.
[0068] 2. Sample DNA extraction process:
[0069] ① DNA extraction reagents for frozen specimens were purchased from Promega (Beijing, China), and the specific steps are as follows:
[0070] Put the tumor tissue into a 1.5mL centrifuge tube containing 600 μL of nuclear lysate and 20 μL of proteinase K, and incubate in a metal bath at 56°C until the tumor tissue is completely digested (no longer than 8 hours). Add 3 μL RNase to the above sample, and incubate in a 36°C metal bath for 15-30 minutes. Add 200 μL of protein sedim...
Embodiment 2
[0151] Example 2 result verification
[0152] A high success rate PCR enzyme KOD FX kit (TOYOBO, Osaka, Japan) was used to perform a PCR experiment to sequence the mutation site of one of the DNA samples of the NK / T cell lymphoma patient in Example 1 to verify the experimental results.
[0153] Specifically, the reaction system of PCR is as shown in Table 12:
[0154] Table 12
[0155] Reagent Dosage Final concentration 2x PCR buffer for KOD FX 25 μL 1× 2mM dNTPs 10μL 0.4mM each 10pmol / μL Primer#1 1.5μL 0.3μM 10pmol / μL Primer#2 1.5μL 0.3μM template DNA 10μL KOD FX (1.0U / μL) 1μL 1.0U / 50μL Autoclaved, distilled water Up to 50μL
[0156] The reaction conditions of PCR are as shown in table 13:
[0157] Table 13
[0158]
[0159] The obtained sequencing results are as figure 1 shown. Will figure 1 The sequencing results shown are consistent with the human genome sequence hg19 in the ucsc databas...
Embodiment 3
[0161] Embodiment 3 Judging prognosis and guiding medication
[0162] 1. Patients with MGA mutations have higher disease stages and poorer prognosis.
[0163] 2. Patients with mutations in genes related to epigenetic acetylation regulation such as EP300, HDAC9, ARID1A are sensitive to targeted drug Chidmide treatment.
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