Optimizing Amplification of the GC-Rich TERT Promoter Region Using 7-Deaza-dGTP for Droplet Digital PCR Quantification of TERT Promoter Mutations

To the Editor: Droplet digital PCR (ddPCR) assays partition PCR reactions into multiple compartments. PCR amplification followed by analysis of end point fluorescence of each partition allows the absolute quantification of the target sequence. ddPCR is effective for rare mutation detection, such as in circulating tumor DNA. Recurrent mutations at 2 sites in the telomerase reverse transcriptase ( TERT )1 promoter are present in multiple malignancies. Two promoter mutations (G>A/C>T transitions occurring at chr5:1295228 or chr5:1295550, respectively, referred to as C228T and C250T) together account for over 80% of TERT promoter mutations in cutaneous melanoma (1). However, the TERT promoter has proved to be a challenging region to amplify due to its high GC content (>80%), homopolymer runs, and low sequence complexity (2). Recently, McEvoy and colleagues reported a ddPCR assay for TERT promoter mutations by use of Q solution and locked nucleic acids (3). We developed an alternative method that uses 7-deaza-2′-deoxyguanosine 5′-triphosphate (7-deaza-dGTP) as an additive for ddPCR. In contrast to McEvoy, our method analyzes the 2 mutations independently. We evaluated the use of dimethyl sulfoxide (DMSO), Q solution, and 7-deaza-dGTP as additives to improve the performance of ddPCR for the detection of TERT promoter …