Microfluidic techniques for DNA melting analysis and digital polymerase chain reaction

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. The platform 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.