The hotspot mutations in codon 132 of isocitrate dehydrogenase 1 (IDH1) is a frequent DNA alteration in gliomas and astrocytomas. IDH mutations are prognostic and diagnostic for tumors with mutation, associated with a proneural subclass, and have longer survival compared with those without the mutation.
We have developed a highly sensitive and simple method to detect hotspot mutations in codon 132 of IDH1 using PNA-mediated real-time PCR clamping, PNAClampTM.
PNAClampTM is based on unique properties of PNA probes that PNA oligomers are not recognized by DNA that can serve as sequence-selective clamps during PCR amplification. This method can also detect 5 mutations through only one tube reactions simultaneously, which requires only 10 ng of the target DNA each.
Data analysis was much faster and easier to perform than analysis of the sequencing data, and results could be obtained in 3hr from DNA extraction to analysis.
Furthermore, we could readily identify a mixture of 1% mutant allele vs. 99% wild type allele with our PNAClamp assay, in comparison to direct sequencing assay which have detection limit of approximately 20% mutant allele.
We performed the assay on 191 paraffin-embedded glioma specimens in Korea for diagnosing IDH mutation.
The results showed that the PNAClampTM IDH1 detected higher number of mutation (27.2%, 52/191) than of direct sequencing (20.9%, 40/191) in 191 giloma samples in Korea. The result was not in agreement with the sequencing in12 (6.3%) samples. This result was likely due to the high sensitivity of PNAClampTM.
PNAClampTM is a fast, sensitive and easy to perform method which can reliably detect common mutations of IDH genes in gliomas or other tumors.
Citation Format: Jihye Yoon, jae jin Choi, Woohyung Choi, Heekyung Park. PNA mediated real-time PCR clamping assay to detect IDH1 mutations . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4225. doi:10.1158/1538-7445.AM2013-4225