Hua Bai1, Da-Jun Deng. 1. Peking University School of Oncology and Beijing Institute for Cancer Research, Beijing 100036, China.
Abstract
OBJECTIVE: To establish a sensitive assay to detect methylation status of the critical 7862nt CpG site related to transcription of HPV16 E6 and E7 genes. METHODS: Genomic DNA of two HPV16-infected cell line SiHa and CaSki was extracted and modified by sodium bisulfite to convert the unmethylated Cs to Us (Ts in PCR products). The target sequence of HPV16 including the 7862nt CpG site was pre-amplified by PCR. Then, the methylation status of the 7862nt site was differentiated in a primer extension reaction with an HPV16-specific primer, and separated by DHPLC at 80 degrees C. RESULTS: The primers without extension and with extension, whether matched to CpG or TpG, could be separated by DHPLC completely. The peak for ddTTP-extension products corresponding to the demethylated CpG site was observed at retention time 6.7 min in both cell lines. However, the peak for ddCTP-extension products representing the methylated CpG site could be detected at retention time 6.3 min in CaSki cell line only, which integrated with 499 methylated and one demethylated HPV16 copies. CONCLUSION: The established DHPLC-primer extension assay can be used to detect methylated and demethylated HPV16 copies simultaneously with a sensitivity up to 1/500 (0.2%).
OBJECTIVE: To establish a sensitive assay to detect methylation status of the critical 7862nt CpG site related to transcription of HPV16 E6 and E7 genes. METHODS: Genomic DNA of two HPV16-infected cell line SiHa and CaSki was extracted and modified by sodium bisulfite to convert the unmethylated Cs to Us (Ts in PCR products). The target sequence of HPV16 including the 7862nt CpG site was pre-amplified by PCR. Then, the methylation status of the 7862nt site was differentiated in a primer extension reaction with an HPV16-specific primer, and separated by DHPLC at 80 degrees C. RESULTS: The primers without extension and with extension, whether matched to CpG or TpG, could be separated by DHPLC completely. The peak for ddTTP-extension products corresponding to the demethylated CpG site was observed at retention time 6.7 min in both cell lines. However, the peak for ddCTP-extension products representing the methylated CpG site could be detected at retention time 6.3 min in CaSki cell line only, which integrated with 499 methylated and one demethylated HPV16 copies. CONCLUSION: The established DHPLC-primer extension assay can be used to detect methylated and demethylated HPV16 copies simultaneously with a sensitivity up to 1/500 (0.2%).