DNA purification increases PCR-amplifiable DNA extracted from formalin-fixed, paraffin-embedded canine mast cell tumors for routine KIT mutation detection

Abstract

DNA amplification by PCR detects KIT exon 11 internal tandem duplications in canine mast cell tumors (MCTs). Tissue-specific inhibitors often contaminate DNA extracted from formalin-fixed, paraffin-embedded (FFPE) canine MCTs, blocking PCR amplification and, consequently, preventing mutation detection. We used a commercial kit to extract DNA from FFPE canine MCTs. Two independent PCR assays, each with one primer set, were used to amplify target genes (HPRT and KIT) directly after FFPE DNA extraction. PCR amplification failed with at least one primer set in 153 of 280 samples (54.6%, 95% CI: 48.8–60.5%). One or 2 DNA washing steps were required to remove PCR inhibitors in 130 of 280 (46.4%) and 23 of 280 (8.2%) of these cases, respectively. DNA concentration and quality (A260/A280 and A260/A230) either pre- or post-washing were not associated with ability of the samples to be amplified by PCR using both HPRT and KIT primer sets. Low-grade and subcutaneous MCTs were less likely to amplify directly after DNA extraction and without any washing steps compared to high-grade MCTs using KIT gene primers.