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 BNAclamp, were used to discriminate the presence of mutant genes mixed with a large number of wild-type genes. Real-time PCR in conjugation with BNAclamping 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 the possibility, 13-mer 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 deference in the Tm values for the 7 BNA bases in the 13-mer 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 oft he mutant and wild-type gene. The effectiveness of blocking the PCR reaction with only 13-mers containing BNA bases allowed us to detect a single mutation, and thus, this BNAclamping real time PCR technology may offer a promising, new avenue to detect clinically importantmutations in the future.
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 BNAclamp, were used to discriminate the presence of mutant genes mixed with a large number of wild-type genes. Real-time PCR in conjugation with BNAclamping 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 the possibility, 13-mer 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 deference in the Tm values for the 7 BNA bases in the 13-mer 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 oft he mutant and wild-type gene. The effectiveness of blocking the PCR reaction with only 13-mers containing BNA bases allowed us to detect a single mutation, and thus, this BNAclamping real time PCR technology may offer a promising, new avenue to detect clinically importantmutations in the future.