Abstract 4753: An effective detection method for the EGFR single mutation T790M using BNA-NC clamping real-time PCR

Abstract

Abstract Mutations on epidermal growth factor receptor (EGFR) cause a variety of cancers including breast and lung cancers. The single mutation T790M on tyrosine kinase domain of EGFR signifies the response to the popular cancer drug gefitinib, which leads to the development of resistance to gefitinib. Detecting the mutation thus guide effective therapeutical options for patients who are in need of cancer drug treatments. We sought to develop a rapid, reliable detection method for the T790M mutation using bridged nucleic acids (BNA), which has been known to enhance the hybridization affinity of oligonucleotides that contain BNA bases. Oligonucleotides containing BNA bases designed to block PCR reaction against wild-type genes, called BNA-clamp, were used to discriminate the presence of mutant genes mixed with a large number of wild-type genes. Real-time PCR in conjugation with BNA-clamping allows us to view the different levels of PCR amplifications in the degree of mixture of wild-type and mutant genes. In an effort to explore this possibility, 13mer long clamps were prepared with various numbers of BNA bases. The clamps containing 9 BNA bases appear to be most effective in blocking the PCR reactions at an optimized concentration to distinguish the mutant from the wild-type genes. In addition to PCR results, the difference in the Tm values for the 7 BNA bases in the 13mer was the largest among differently designed clamps, which is consistent with the results obtained by real-time PCR. We also examined the degree of sensitivity using the clamp containing 9 BNA bases, revealing that the clamp has the ability to determine the level of mutation as low as 1% mixture of the mutant and wild-type gene. The effectiveness of blocking the PCR reaction with only 13mers containing BNA bases allowed us to detect a single mutation, and thus, this BNA-clamping real time PCR technology may offer a promising, new avenue to detect clinically important mutations in the future. Citation Format: Sung-Kun Kim, Xiaoyun Liu, Aaron Castro, Leticia Loredo, Miguel Castro. An effective detection method for the EGFR single mutation T790M using BNA-NC clamping real-time PCR. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4753. doi:10.1158/1538-7445.AM2015-4753