Establishment And Application Of An Effective Method Based On Real Time Fluorescence Quenching For Single Nucleotide Polymorphism Detection

Currently, single nucleotide polymorphism （SNP） analysis has become an important means to determine the genetic disease and individual drug response characteristics, but also an essential means of prediction and prevention of many diseases. Methods for SNP detection, such as sequencing and TaqMan platform, have some drawbacks, however, we described an effective method based on real time fluorescence quenching for SNP detection. The Quenching-PCR platform used a probe with quencher to eliminate fluorophor of the terminal base according to dideoxy sequencing method. Compared with the consequences of Sanger’s sequencing, the results of Quenching-PCR showed a high concordance rate （93.40%）, while the results of TaqMan platform showed a concordance rate of 92.45%, indicating that Quenching-PCR and TaqMan assay were similar in accuracy. Considering cost, time and probe design, Quenching-PCR was more suitable as a clinical SNP method. Therefore, Quenching-PCR will be easily applicable and has great effect on clinical diagnosis.We aimed to detect related genes of personalized medicine by SNP detection method based on fluorescence quenching. The loci related tyrosine kinase inhibitor therapy is 2235₂249 on exonl9 and L858R on exon21 of epidermal growth factor receptor （EGFR）. The loci related platinum drug are locus of Val417Ile on gene multidrug resistance-associated protein 2 （MRP2）, locus rs3212986 on gene excision repair cross-complementation group 1 （ERCC1） and locus Arg194Trp on X-rayrepaircrosscomplementingl （XRCC1）. We used Quenching PCR to detect common loci related genes of personalized medicine in oncology, in order to validate the Quenching PCR. At the same time, we had showed that Quenching-PCR could also be used in Insert/Delete （InDel） detection.Tumor heterogeneity is closely related to tumor metastasis, and has become a focus of tumor research in recent years. Colon carcinoma is a common malignant tumor with high heterogeneity. Differences in morphology, immune phenotype and molecular genetics have led to different reactions and prognosis for the clinical treatment of colon carcinoma.There are differences in tumor heterogeneity between individuals, while there are differences in genomes between separate regions and metastasis. This study has detected genome-wide SNPs in tumor tissue, adjacent tissue and metastases of colon carcinoma patients. We aimed to find whether heterogeneity is related to disease progression and metastasis of tumors by bioinformatics analysis. We aimed to study the correlation of heterogeneity of primary and metastasis of colon carcinoma by SNP detection method based on fluorescence quenching. The change of copy number variation （CNV） in metastases was more significant than that in adjacent tissues of tumor and primary tumors （p< 0.01）. The differential genes between metastases and primary tumors were clustered, and 1/4 of the related pathways belonged to adhesion and immunity. Related genes of differential SNPs in encoding region between metastases and primary tumors were clustered,1/2 of GO categories belonged to metastasis and immunity categories while 1/5 of the related pathways belonged to adhesion and immunity. Similarly, related genes predicted by differential miRNAs between metastases and primary tumors were clustered, and 1/3 of the related pathways belonged to metastasis and immunity while 1/5 of the related pathways belonged to adhesion and immunity. These results indicated that adhesion and immune regulation may be essential for the development of tumor. Differential SNPs of chips were analyzed by comparing the results of metastases and primary tumors, and more samples were validated by Quenching-PCR. The locus rs 12881063 in the fourteenth chromosome was found to have a high mutation rate in metastases. The mutation rate reached 66.7% in nearby metastases, and the mutation rate reached 83.3% in the distant metastases. These results suggest that the mutation of the locus may be associated with the metastasis of tumor, which may be the basis for the clinical diagnosis of tumor.