Reduction of Sequence Artifact in Formalin Fixed Paraffin Embedded Tissue Using Uracil DNA Glycosylase: A Comparative PCR High Resolution Melting Curve Analysis
J Anthony Halfacre, James M Gale, Mohammad A Vasef. University of New Mexico, Albuquerque, NM; TriCore Reference Laboratories, Albuquerque, NM
Background: Sequence artifact is occasionally detected in DNA obtained from formalin fixed paraffin embedded (FFPE) archival tissue. The sequence artifacts include C>T/G>A and is thought to be due to cytosine deamination to uracil. Although the sequence artifacts in DNA from FFPE tissue is not reproducible, it might potentially lead to errors including false positive results when using high resolution melting (HRM) curve assays for detection of mutations in genes such as KRAS, BRAF, and EGFR to determine efficacy of targeted therapy. In this study, we examined the effects of uracil DNA glycosylate (UDG) treatment in reducing C>T/G>A sequence artifacts.
Design: DNA extracted from FFPE tissue sections of histologically documented lung adenocarcinoma (51 samples) and gastrointestinal stromal tumors (3 samples) were examined for exons 18, 19, 20, and 21 of EGFR and exons 9 and 11 of GIST cases respectively with and without UDG incubation prior to parallel PCR amplification. For UDG treatment, the DNA samples were incubated with this enzyme at 37 degree Celsius for 30 minutes before the PCR amplification. Next, the PCR amplified products were melted using high resolution melt (HRM) technique (Idaho Technology). The melting curves of UDG treated and UDG untreated samples were then compared with melting curves of known positive and negative controls.
Results: Wild type (WT) samples with severe shouldering artifact that raised the possibility of mutations were identified in approximately 5% of DNA samples without UDG treatment. However, the shouldering artifact was not present on melting curves of the parallel samples that had been treated with UDG as illustrated in Figure 1.
Conclusions: Incubation of DNA extracted from FFPE tissue with UDG significantly improve the shouldering artifact on melting curve assays used for mutational analysis of genes with available targeted therapy such as KRAS, BRAF, KIT, and EGFR. Treatment of DNA with UDG prior to amplification and melting analysis improve sequence artifact and reduces potential false positive results.
Monday, March 4, 2013 9:30 AM
Poster Session I Stowell-Orbison/Surgical Pathology/Autopsy Awards Poster Session # 307, Monday Morning