Application of KAZN gene methylation detection reagent in preparation of colorectal cancer prognosis diagnosis reagent
A technology for genetic testing and diagnostic reagents, applied in the field of genetic diagnosis, can solve the problems of lack of coverage and limited value of molecular markers
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Embodiment 1
[0093] Embodiment 1 sample source
[0094] Case Sample Patient Characteristics
[0095]Patients with pathologically verified stage I-II CRC who underwent surgical resection were included as cases in the discovery, training, or validation cohorts. Patients who had previously received any anticancer therapy, a history of any tumor other than CRC, and patients with extensive degradation of DNA samples were excluded.
[0096] First, 45 fresh-frozen tumor tissues and adjacent normal tissues were collected from patients with stage I-II CRC for genome-wide methylation microarray analysis. Patients with intestinal obstruction or perforation, vascular or neural invasion, and fewer than 12 lymphadenectomy submissions were excluded. Based on age, sex, TNM stage, date of surgery (±5 years), and tumor location, this cohort of 45 patients included 21 patients with recurrence at follow-up and 24 patients who achieved tumor recurrence-free survival at follow-up. These 45 patients constitut...
Embodiment 2
[0103] Example 2 Methylation detection of KAZN gene
[0104] qMSP was used to detect the methylation level of gene CpG sites.
[0105] Gene detected: KAZN;
[0106] Comparison genes: FHIT, SGIP1.
[0107] 1. Quantitative methylation-specific PCR
[0108] Genomic DNA was extracted and modified with bisulfite using QIAamp DNA Mini Kit (Qiagen, 51306) and EZ DNA Methylation Kit (ZymoResearch, D5002).
[0109] Quantitative methylation-specific PCR (qMSP) was used to detect the tested CpG sites located in the gene body or CpG open sea in different cohorts to evaluate and verify their relationship with the prognosis of CRC patients.
[0110] In this assay, bisulfite-converted genomic DNA is amplified using primers and a pair of oligonucleotide probes covering the CpG sites to be tested, each oligonucleotide probe is attached to the 5' end of a fluorescent reporter The dye 6FAM or VIC (specifically binding to methylated sites and unmethylated sites, respectively) is coupled with ...
Embodiment 3
[0155] Example 3 Detection Results of KAZN Gene Methylation Degree in Tumor Tissues of Patients with Colorectal Cancer
[0156] As shown in Table 4 below, in the tumor tissues of 8 patients with stage I-II colorectal cancer, the methylation degree of KAZN gene was detected according to the method of Example 2, and the methylation ratio was higher than or equal to 66.39%, which was judged as hypermethylation Methylation, less than 66.39% was judged as hypomethylation. From the results in Table 4, it can be seen that the recurrence risk of colorectal cancer patients with high methylation of KAZN was significantly higher than that of patients with low methylation.
[0157] Table 4 Detection results of KAZN gene methylation degree
[0158]
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