Personalized Clinical Measurement Of Rare Mutations Using Xenodna: Preliminary Application For Early Treatment Response And Screening

Though DNA repair is an efficient process, a mutation occurs for every 1/2,000 to 1/20,000 single-strand breaks. Because these are primarily point and indel mutations that are difficult to detect, the potential for measuring mutational burden after a genotoxic event is daunting. Nevertheless, expensive and complex new polymerases, faster and more accurate sequencing technologies, and mathematical estimators have made it possible to achieve improved detection of mutations. A simpler method to quantitatively measure the mutations in an overwhelming background of wide-type genes with greater sensitivity and accuracy would have enormous opportunities in biology and medicine. We will describe QClamp, a method that can be adapted to most existing technologies, adding an additional >1,000-fold improvement in point and indel mutation detection. QClamps are easily designed and synthesized xenoDNA that prevent PCR amplification of the wild-type sequence, allowing only the amplification of mutations. The method employs aminoglycol linkers between the DNA bases to replace the usual phosphate backbone. The charge and stiffness of this structure allows for better binding to wild type than the normal consensus sequence but also a ≈30°C decrease in melting temperature for even one point mutation. Thus, only the wild type is blocked from replication during PCR amplification. The QClamps can be implemented into any polymerase reaction and incorporated into most technologies, including PCR, NGS, and CRISPR. As proof of principle, we will show: 1. Quantitative detection of 1:100,000 mutant: wild type using ordinary PCR reagents. 2. Off-the-shelf QClamps for most cancer mutations, allowing for personalized liquid biopsy biomarker creation. 3. Real-time documentation of clinical response using quantitative plasma DNA detection within 24 hr of treatment initiation. 4. Improved screening potential with >95% of carcinomas and >60% of adenomatous polyps detectable in the stool. QClamp may help achieve the full potential of liquid biopsy for personalized cancer screening and economical and efficient measurement of tumor response mid-treatment. The latter application should be useful for patients with subclinical disease, patients receiving adjuvant radiation or chemotherapy for high-risk disease, and patients needing early indication of response following initiation of a chemotherapy regiment.