UC Berkeley

Novel methodologies for the molecular detection of human cancer have been advanced using microfabricated capillary electrophoresis devices as analytical platforms. These techniques enable direct and quantitative characterization of the unique biomolecular signature inherent to individual cancers and, through reduction of sample usage and analysis time, further the goal of routine genetic screening in the clinical setting.

The majority of the work detailed here applies a comparative sequencing technique known as Polymorphism Ratio Sequencing (PRS) to cancer detection with a focus on mutations in the mitochondrial DNA (mtDNA). First, a rigorous optimization of sample processing protocols was undertaken to improve PRS separations on a 96-lane microfabricated sequencing device. A modification of electrokinetic injection conditions has increased capillary success rates to nearly 100%, while the introduction of dynamic coatings and automated data processing has decreased analysis time by 75%. These optimized conditions were validated through a complete mtDNA sequence comparison of two unrelated individuals, uncovering 44 confirmed germline variations, eight of which were undetected in early PRS experiments. Further analysis of paired tumor and blood mtDNA from six individuals with lung and bladder cancer revealed three heteroplasmic somatic variants while uncovering 18 erroneous mutations identified in previous microarray analysis.

To establish potential clinical relevance, PRS was independently applied to a mitochondrial D-loop analysis of fourteen bladder cancer patients. A total of 21 somatic variations were identified, with seven patients harboring at least one mutation. Fifteen of these mutations were heteroplasmic, often occurring at low levels or problematic base locations inaccessible to conventional technologies. Where available, matched urine mtDNA was found to contain abundant populations of the mutant genotype, establishing the potential use of bodily fluids for noninvasive screening.

Finally, an integrated microdevice capable of PRS extension followed by inline purification and electrophoretic separation is presented. This device makes use of dual on-chip thermal cyclers and orthogonal Sanger extension primers to generate a complete set of PRS fragments prior to oligonucleotide-based capture and injection. Further integration with upstream sample processing steps, including single-cell capture and PCR amplification, is proposed, providing the framework for real-time mutant quantitation in microbiopsies, ultimately enabling full clinical integration.