Sensitive and Specific KRASSomatic Mutation Analysis on Whole-Genome Amplified DNA from Archival Tissues

Kirsten RAS (KRAS) is a small GTPase that plays a key role in Ras/mitogen-activated protein kinase signaling; somatic mutations in KRASare frequently found in many cancers. The most common KRASmutations result in a constitutively active protein. Accurate detection of KRASmutations is pivotal to the molecular diagnosis of cancer and may guide proper treatment selection. Here, we describe a two-step KRASmutation screening protocol that combines whole-genome amplification (WGA), high-resolution melting analysis (HRM) as a prescreen method for mutation carrying samples, and direct Sanger sequencing of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue, from which limited amounts of DNA are available. We developed target-specific primers, thereby avoiding amplification of homologous KRASsequences. The addition of herring sperm DNA facilitated WGA in DNA samples isolated from as few as 100 cells. KRASmutation screening using high-resolution melting analysis on wgaDNA from formalin-fixed, paraffin-embedded tissue is highly sensitive and specific; additionally, this method is feasible for screening of clinical specimens, as illustrated by our analysis of pancreatic cancers. Furthermore, PCR on wgaDNA does not introduce genotypic changes, as opposed to unamplified genomic DNA. This method can, after validation, be applied to virtually any potentially mutated region in the genome.