A gene associated with tumor recurrence after liver cancer surgery and its detection method
A liver cancer and reagent technology, applied in the field of molecular biology, can solve problems such as unclear mechanism and unclear gene changes
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0031] Example 1. Detection of MPPE1 Gene Point Mutations by PCR–Sanger Sequencing
[0032] 1. DNA extraction from cancer tissue
[0033] Using the QIAamp DNA Kit kit to extract tissue genomic DNA, specific operations:
[0034] ①Wash the mortar, oak grinder and homogenizer and other utensils, rinse with DEPC water, and dry at 200°C for 4 hours to remove RNase;
[0035] ② After the high-temperature sterilized vessel is cooled at room temperature, add liquid nitrogen and use a tool to pre-cool it, quickly take out the tissue from the liquid nitrogen, cut out about 50-100mg in size, and quickly grind it in a mortar;
[0036] ③ Transfer the ground tissue fragments to an RNase-free centrifuge tube added in 180 μl buffer ATL in advance, and mix well;
[0037] ④Add 20μl 1.25 proteinase K and mix thoroughly. 56℃ water bath, overnight;
[0038] ⑤Take it out, quickly shake and mix, add 200μl buffer AL, mix well; 70℃ water bath, 10min
[0039] ⑥Centrifuge at 8000rpm for 2 minutes, a...
Embodiment 2
[0058] Embodiment 2, detection of MPPE1 gene point mutation by PCR-MassArray mass spectrometry
[0059] Identify whether a C / T point mutation has occurred at position 11897016 of the MPPE1 gene located on chromosome 18, that is, identify whether the C / T point mutation has occurred at position 49 of the amplified product. 2. PCR-MassArray mass spectrometry for XX gene Detection of point mutations
[0060] 1. Experimental design and primer synthesis: The company is responsible for the design and synthesis, and then the quality of the primers and the adjustment of the primer reaction concentration. The position of the primers in the target sequence as figure 2 shown.
[0061] 2. Experimental process
[0062] 1) PCR amplification:
[0063] PCR reaction system: 5ul (4ul mix+1ul DNA)
[0064] Table 2. PCR reaction system in PCR-MassArray mass spectrometry
[0065]
[0066]
[0067] *MgCl 2 The final concentration is 4.0mM, 2.0mM comes from the PCR buffer, 2.0mM comes f...
Embodiment 3
[0099] Embodiment three, multi-sample detection
[0100] The detection of 164 cases of clinical samples: including 86 cases of hepatitis B-related liver cancer tissues, 35 cases of hepatitis B-related liver cancer tissues after liver transplantation, and 43 cases of benign liver disease tissues (control). Through advanced biological information analysis technology and statistical methods, combined with preoperative clinical data and pathological characteristics of samples (age, gender, TNM stage, tumor size, number, distribution, vascular invasion, lymph node metastasis, histological differentiation, serum AFP, etc.) and their corresponding recurrence, metastasis, and prognosis, the C / T point mutation detection of the 11897016th site of the MPPE1 gene, and the detection of the p53 gene mutation were performed to compare and analyze the relationship between the mutated gene and the patient's clinicopathological characteristics and prognosis. Correlation. In the prior art, the ...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More