The mutant sequence, detection method, specific primer pair used and application of the adgrg6 enhancer
A technology for detecting primers and specificity, applied in biochemical equipment and methods, microbe determination/testing, DNA/RNA fragments, etc., to achieve the effect of high density of new blood vessels
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Embodiment 1
[0046] Example 1: Sample collection and whole genome sequencing analysis:
[0047] In accordance with medical ethics and with the patient's consent, 65 pairs of paired bladder tumor tissue and peripheral blood samples were obtained from newly diagnosed individuals with bladder cancer in the Institute of Urology, Shenzhen University for whole genome sequencing analysis of 65 cases of bladder cancer The clinical information of the patients is shown in Table 1.
[0048] Table 1 Clinical information table of 65 patients with bladder cancer analyzed by whole genome sequencing
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[0052] Genomic DNA was extracted from 65 bladder cancer patients shown in Table 1 for whole-genome sequencing. The specific method is as follows:
[0053] 1. Raw data was filtered using SOAPnuke (v.1.5) to remove adapter sequences and low-quality reads. The high-quality reads were aligned with the human reference genome (hg19, NCBI GRCh37) by Burrows-Wheeler Aligner (...
Embodiment 2
[0059] Example 2: Validation of ADGRG6 enhancer mutations:
[0060] In the case of conforming to medical ethics, 196 samples of bladder cancer who had not undergone radiotherapy and chemotherapy were screened from the paraffin sections of bladder cancer preserved in the sample bank of the Institute of Urology, Shenzhen University. The information of the 196 samples is shown in Table 2.
[0061] Table 2 Clinical information table of 196 patients with bladder cancer targeted verification
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[0067] In order to further verify the mutation of the ADGRG6 gene enhancer in bladder cancer, specific primers targeting the mutation region of the ADGRG6 enhancer were designed, and targeted verification was performed in 196 newly enrolled bladder cancer samples by Sanger sequencing. The specific operation process is:
[0068] DNA was extracted from the 196 samples shown in Table 2 using the TIANamp FFPE DNA kit (Tiangen Biotech),...
Embodiment 3
[0074] Example 3: Effects of ADGRG6 enhancer mutations on ADGRG6 expression and microangiogenesis in tumor tissues:
[0075] In order to further explore the effect of ADGRG6 gene enhancer mutation on tumor in bladder cancer, the expression of the protein encoded by ADGRG6 gene in tumor tissue was analyzed by immunohistochemical method, and based on the biological function of ADGRG6 to promote angiogenesis, The effect of ADGRG6 enhancer mutations on blood vessel density in tumor tissues was analyzed. Specific steps are as follows:
[0076] In 196 cases of bladder cancer tumor samples with confirmed ADGRG6 enhancer mutations, the expression of ADGRG6 protein in ADGRG6 enhancer mutation and wild-type samples and the formation of microvessels in tumor tissues were analyzed by immunohistochemical methods.
[0077] The number of microvessels was assessed by immunohistochemistry for CD31. Using specific antibodies against ADGRG6 (Abcam, catalog number ab117092) and anti-CD31 (Abc...
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