Erroneous identification of APOBEC3-edited chromosomal DNA in cancer genomics

Background: The revolution in cancer genomics shows that the dominant mutations are CG- > TA transitions. The sources of these mutations are probably two host cell cytidine deaminases APOBEC3A and APOBEC3B. The former in particular can access nuclear DNA and monotonously introduce phenomenal numbers of C- > T mutations in the signature 5'TpC context. These can be copied as G- > A transitions in the 5'GpA context. Methods: DNA hypermutated by an APOBEC3 enzyme can be recovered by a technique called 3DPCR, which stands for differential DNA denaturation PCR. This method exploits the fact that APOBEC3-edited DNA is richer in A+T compared with the reference. We explore explicitly 3DPCR error using cloned DNA. Results: Here we show that the technique has a higher error rate compared with standard PCR and can generate DNA strands containing both C- > T and G- > A mutations in a 5'GpCpR context. Sequences with similar traits have been recovered from human tumour DNA using 3DPCR. Conclusions: Differential DNA denaturation PCR cannot be used to identify fixed C- > T transitions in cancer genomes. Presently, the overall mutation frequency is similar to 10(4)-10(5) base substitutions per cancer genome, or 0.003-0.03 kb(-1). By contrast, the 3DPCR error rate is of the order of 4-20 kb(-1) owing to constant selection for AT DNA and PCR-mediated recombination. Accordingly, sequences recovered by 3DPCR harbouring mixed C- > T and G- > A mutations associated with the 5'GpC represent artefacts.