| Publication Type |
dissertation |
| School or College |
College of Engineering |
| Department |
Biomedical Engineering |
| Author |
Sundberg, Scott Owen |
| Title |
Microfluidic techniques for DNA melting analysis and digital polymerase chain reaction |
| Date |
2010-08 |
| Description |
Microfluidic methods were applied to nucleic acid mutation identification and quantification. DNA melting analysis interrogation volumes were reduced 4 orders of magnitude (down to 1 nL volumes) from commercial instrumentation, allowing less reagent consumption while yielding adequate signal for genotyping and scanning of polymerase chain reaction (PCR) products. A microfluidic instrument was developed for digital PCR applications, using a spinning plastic disk patterned by xurography. Theplatform offers faster thermocycling times (30 cycles in ~12 min), simplified fluid partitioning, and a less expensive disposable when compared to currently available digital PCR platforms. PCR within the disk was validated by quantifying plasmid DNA sample using "on/off" fluorescence interrogation across 1000 wells (30 nL/well) at varying template concentration. A 94% PCR efficiency and product amplification specificity were determined by aggregate real-time PCR and melting analysis. The technique of quasi-digital PCR was also applied within this platform, wherein a single mutation copy was preferentially amplified from a large background of wild-type DNA, to detect and quantify low levels of rare mutations. This method demonstrated a sensitivity of 0.01% (detecting a mutant to wild-type DNA ratio of 43:450000), by mixing known concentrations of an oncogene mutation with thousands of wild-type template copies. Statistic analysis tools were constructed in order to interpret digital PCR data, with results comparing well to DNA absorption measurements. |
| Type |
Text |
| Publisher |
University of Utah |
| Subject |
Circulating tumor cells; Digital PCR; DNA melting analysis; Microfluidics; PCR |
| Dissertation Institution |
University of Utah |
| Dissertation Name |
Doctor of Philosophy |
| Language |
eng |
| Rights Management |
Copyright © Scott Owen Sundberg 2010 |
| Format Medium |
application/pdf |
| Format Extent |
15,745,599 bytes |
| Source |
Original in Marriott Library Special Collections, QH9.7 2010 .S86 |
| ARK |
ark:/87278/s60k2qb9 |
| Setname |
ir_etd |
| ID |
194712 |
| Reference URL |
https://collections.lib.utah.edu/ark:/87278/s60k2qb9 |
