A Rapid Colorimetric Peptide Nucleic Acid Loop-Mediated Isothermal Amplification Assay for the Detection of the IDH1 Mutation in Glioblastoma

<p>The <em>IDH1</em> mutation is an important diagnostic and prognostic biomarker used to characterize glioblastoma (GBM). Patients harboring the <em>IDH1</em> mutation have improved overall survival following maximal resection. Knowledge of the <em>IDH1</em> mutation status allows the surgeon to modify the surgical plan; however, no existing molecular test can provide this information intraoperatively. We designed a novel colorimetric peptide nucleic acid loop-mediated isothermal amplification (PNA-LAMP) method that rapidly detects the <em>IDH1</em> R132H mutation in GBM. PNA-LAMP amplifies target DNA under isothermal conditions with high specificity and speed. The PNA prevents amplification of wild-type <em>IDH1</em> DNA, while allowing amplification of the R132H variant if present. We used a pH-sensitive colorimetric detection method for visual determination of amplification in under one hour. Characterization of the assay was performed with plasmid DNA containing the <em>IDH1</em> wild-type and R132H variant sequences. Amplification was confirmed using gel electrophoresis, and this analysis suggests that the assay is more sensitive than Sanger sequencing – the gold standard for <em>IDH1</em> mutation identification. This study is the first to attempt to develop a colorimetric LAMP assay for GBM tumor characterization, and only the third application of the PNA-LAMP method to detect acquired mutations in cancer. This novel molecular assay is a simple, specific, and rapid way to identify the presence of the <em>IDH1</em> R132H variant associated with GBM. </p>

Name thesis:degree_name

Master of Science

Level thesis:degree_level

Thesis

Discipline thesis:degree_discipline

Clinical Sciences

Year dc:date.available

2017

Author dc:creator

• Raack, Edward J

Contributors dc:contributor

• Dr. Paul Mann

• Loop-mediated isothermal amplification
• glioblastoma
• isocitrate dehydrogenase
• molecular genetics
• assay development
• Biotechnology
• Genetics
• Genetics and Genomics

Repository record dc:identifier

https://commons.nmu.edu/theses/158

OAI identifier oai:identifier

oai:commons.nmu.edu:theses-1173