An ultra-sensitive method to detect mutations in human templates.

The RAS family of small GTPases is mutated in roughly a fifth of human cancers. Hotspot point mutations at codons G, G, and Q account for 95% of all these mutations, which are well established to render the encoded proteins oncogenic. In humans, this family comprises three genes: , and . Accumulating evidence argues that oncogenic RAS point mutations may be initiating, as they are often truncal in human tumours and capable of inducing tumorigenesis in mice. As such, there is great interest in detecting oncogenic mutation in the RAS genes to understand the origins of cancer, as well as for early detection purposes. To this end, we previously adapted the microbial ultra-sensitive aximum epth equencing (MDS) assay for the murine gene, which was capable of detecting oncogenic mutations in the tissues of mice days after carcinogen exposure, essentially capturing the very first step in tumour initiation. Given this, we report here the adaption and details of this assay to detect mutations in a human sequence at an analytic sensitivity of one mutation in a million independently barcoded templates. This humanized version of MDS can thus be exploited to detect oncogenic mutations in at an incredible sensitivity and modified for the same purpose for the other RAS genes.