A Mutation Sensitive Switch Assay To Detect Five Clinically Significant EGFR Mutations

Objective: To develop methods to detect five common EGFR somatic mutations in tumor tissues from NSCLC patients by using a nanoscale mutation-sensitive switch consisting of a high fidelity polymerase and phosphorothioate-modified allele-specific primers.Methods: Mutant templates and wild type templates were obtained from either regular PCR using Taq DNA polymerase or overlap extension PCR. The PCR products were gel purified and sub-cloned into the PMD19-T vector. Cloning products harboring both normal and mutant EGFR fragments were confirmed by direct sequencing. Blood samples were obtained from normal controls, and tissue samples were from lung cancer patients. The five clinically significant EGFR mutations examined here are: S768 I, T790 M,L858R and 15-bp and 18-bp deletion mutations. Five pairs of primers were used to detect the five mutations. The forward primers had a 3’ terminal phosphorothioate modification while the common reverse primer had no specific modification. A nanoscale proofreading polymerase-mediated on/off switch was employed in these five loci to detect the S768 I,T790M and L858 R point mutations as well as the 15-bp(E746-A750) and 18-bp(L747–S752) deletion mutants in exon 19. The molarity of the wild type and mutant templates was adjusted to 1:1 and then diluted to 107, 106, 105, 104, 103, 102 and 101copies/μL to test the sensitivity of the phosphorothioate modification and high fidelity enzyme. Rear-time PCR and multiplex PCR were also used in this assay.Results: The wild type fragments for S768 I, T790 M, L858 R and exon 19 were successfully inserted into the recominant plasmids, and the mutant templates for S768 I,T790M, L858 R, 15-bp deletion(E746-A750) and 18-bp deletion(L747–S752) were also successfully prepared. The major components of these specific assays include high fidelity polymerases with intrinsic proofreading activity and nuclease-resistant specific primers targeting the wild type sequence and mutant plasmids harboring S768 I, T790 M, L858 R and the 15-and 18-bp deletions. Five assays were successfully developed for each of theselected mutations. The allele-specific primers for the S768 I, T790 M, L858 R, 15- and18-bp deletion mutations could detect the perfectly matched template at a sensitivity of about 100 copies. The same assay conditions yielded no primer extension products until template levals met or exceeded 105 copies of mismatched amplicon. The assays to detect the S768 I, T790 M, L858 R, 15- and 18-bp deletion mutations are highly sensitive and can detect as few as 10-100 template copies. These assays are also sufficiently specific so that mismatched primers are not extended until the number of template copies exceeds 105. The accuracy and sensitivity of fluorescence quantitative PCR is higher than ordinary PCR. The real-time PCR had weak amplification with 105 copies for the wild type template, which is similar to the gel electrophoresis results. Similar results were obtained for multiplex PCR for S768 F, S790 F, S858 F and the exon 19 15/18-bp deletions. The multiplex PCR was used to detect the S768 I, T790 M and L858 R point mutations as well as the exon 19 15- and18-bp deletions in tumor samples. In screening DNA samples from these 20 NSCLC cases,two cases with S858 F mutations were identified. The results from a sequencing analysis were consistent with these experimental results.Conclusion: In this study a high fidelity DNA polymerase-mediated mutation sensitive switch was applied to analyze five clinically significant EGFR mutations. The results showed that the mutation-sensitive switch is sufficiently sensitive and specific to detect these mutations in both plasmid samples and DNA from tumor samples. This new assay is easy to perform and economical, and can also be combined with other platforms such as real-time PCR and multiplex PCR for detecting mutations. The on/off switch assay based methods showed reliable, accurate and reproducible results in the detection of these five EGFR mutations. Thus, they may have potential use for monitoring EGFR-targeted therapies.