Investigation of Ex vivo detection of DNA adducts in relation to mutation frequency

Mutations in human genes have been extensively identified and studied. Many genes show characteristic ‘Hot Spots’ for mutations and mutational spectra. The former often cause oncogenic changes that drive cancers, whilst the latter reflects prevailing sources of DNA damage that lead to mutation. Many types of DNA damaging agents and DNA repair mechanisms are known but their significance at the level of an individual base sequence is unclear and methods to determine specific positions of DNA damage in vivo are not available. This means that the processes that determine overall susceptibility to damage and how it leads to fixation as a deleterious mutation and is subsequently selected cannot be fully understood. This is exemplified by the human genome RAS genes. They have highly similar sequences. All three isoforms become oncogenic when constitutively activating mutations occur in either of two mutational “Hot Spots”, codons 12 to 13 and codon 61. Despite these similarities, they have very different mutational spectra. Here, the prospect of developing a non-a priori method for detecting sequence specific damage ex vivo was investigated. Methods of sequence target enrichment are DNA damaging by their nature because they require heat and heat is DNA damaging. Isothermal methods are needed. It is shown here that a loop oligonucleotide ligated to both ends of a DNA fragment allowed it to be copied in a highly repetitive fashion by Rolling Circle Amplification and the products could be read as single molecules by a next generation sequencer. The prospect for using the RecA protein for isothermal target enrichment was also demonstrated. Sequence accuracy was explored in the presence of new and known DNA damage. It was found that features of the sequencer used, primarily its reliance on single stranded DNA from a double strand, limited the accuracy of sequencing for the purpose here. Further work based on these investigations is expected to deliver the required method, allowing unprecedent measurements of DNA damage at specific sequences