Application Of Mutation-sensitive Molecular Switch In The Detection Of EGFR Mutations Associated With Lung Cancer

Aim: To study the application of the mutation-sensitive molecular switch in the two deletion hotspots of EGFR gene associated with lung cancer.Methods: Two hotspots deletions in exon 19 of EGFR gene were chosen as the detection targets of this research project. They are 15 nt deletion of E746–A750(del 2235–2249) and 18 nt deletion of L747–P753 insS(del 2240–2257). Allelic specific primers targeting wild type and mutation type were desgined with the primers'3'terminal phosphorothioate modification. A large amount of positive control templates of wild type and deletion mutations were prepared using cloned plasmids containing relevant DNA fragments respectively. Genomic DNAs were extracted from blood samples donated by healthy volunteers, from pathologically"normal"lung tissue, and from lung cancer tissue. Within a broad range of annealing temperature from 48 to 68°C, the 3'phosphorothioate-modified allelic primers were compared with those without modification, and the allelic specific amplifications by mutation-sensitive molecular switch using Pfu polymerase were compared with Taq polymerase. DNA samples from lung tissue of 6 lung cancer patients were analyzed. The assays for EGFR deletion detection were evaluated for their compatibility in real time PCR platform, with which the sensitivities and specificities of the assays were determined.Results: Two assays for EGFR 15 nt and 18 nt deletion mutations analysis have been developed with the mutation-sensitive molecular switch. Within a broad range of annealing temperature, mutation-specifci molecular switch showed"on"effect on mutation templates and"off"effect on wild type DNA templates. The"off"effect, or the specificity of the assays was annealing temperature dependent: higher annealing temperature displayed higher specificity in mutation discrimination. The wild type templates and mutation templates of plasmids were serially diluted from 10~8,10~7,10~6,10~5,10~4,10~3,10~2,to 10~1 copies/μl. While annealing-temperature-dependent false positives were observed in controls amplified with Taq together with phosphorothioate-modified primers or Pfu together with unmodified allelic specific primers, mutation-sensitive molecular switch was able to detect 100 copies for the deletion mutation. The regression analysis between copy number and cycle of threshold 1/2 displayed good linear relationship for the 15 nt deletion mutation. These standard curves from real time PCR data had coefficiecy of 0.990。In screening DNA samples from 6 cases of lung cancer, one 15bp deletion was identified. Interestingly, routing sequencing analysis is not sensitive enough to identify the mutation in this case. Probably due to the mutated lung cancer is the small portion of the cancer tissue or the biopse sample, the mutated signal could not be read by routine sequencing analysis.Conclusion: In addition to point mutation analysis, the high fidelity DNA polymerase-mediated mutation-sensitive molecular switch is applicable in deletion assay. Furthermore, the two assays of 15 nt deletion and 18 nt deletion in EGFR gene are compatible with real time PCR, which enables the sensitive analysis for somatic rare mutation.