Exploring Taq polymerase induced mutations in part of BRAF exon 15 by sequencing and mutation enrichment

The polymerase chain reaction (PCR) is a fundamental technique in molecular biology, enabling the amplification of specific DNA sequences for various applications. Despite its utility, the fidelity of DNA polymerases used during amplification is not perfect, leading to the introduction of replication errors such as base substitutions, insertions, and deletions into the amplified products. These errors can significantly impact downstream analyses, where correct identification of true biological DNA variations is crucial. This study aimed to investigate the error rates of Taq polymerase during PCR amplification of a 127 base pair sequence surrounding codon 600 of the BRAF gene. This region, located in exon 15, is of significant clinical interest due to its association with various cancers. Using the MiSeq platform in combination with a constant denaturant capillary electrophoresis (CDCE) assay, we analyzed the errors introduced by Taq polymerase. On average, 1.1 million sequencing reads were generated for each PCR-amplified sample, in combination with mutant enrichment through CDCE to allow for the observation of mutations from the background noise. We identified a non-random mutational spectrum with respect to positions, with several hotspots. The majority of mutational changes were transitions, either A to G or T to C. Noteworthy is the mutation in codon 600, where the second base “T” is mutated to an “A,” corresponding to the clinically relevant V600E mutation. This study highlights the importance of understanding polymerase-induced errors in PCR and their implications for NGS data accuracy. The enrichment strategy employed here demonstrates a method for detecting and analyzing these errors, providing valuable insights for genomic research and clinical applications. • Taq polymerase generated a non-random mutational spectrum in BRAF exon 15 , including recurrent hotspots detectable only after CDCE-based enrichment. • Clinically relevant V600E (T→A) mutations arose as polymerase-induced artefacts , demonstrating that Taq can generate disease-associated variants under standard PCR conditions. • CDCE enrichment combined with deep MiSeq sequencing enabled detection of low-frequency polymerase errors that remained invisible in unenriched libraries. • Findings highlight the risk of false-positive mutation calls in low-template or high-cycle assays , emphasizing the need for proofreading enzymes in clinical and sensitive analyses.