Doctor of Philosophy (PhD)
Recognition of point mutations in a codon 12 of the K-ras gene, most frequently observed, is considered to be useful in the early diagnosis of several types of the human cancers. We have developed a multifunction, disposable, microfluidic module which detects low-abundant point mutations in human genomic DNA in modular architecture. Each functional component including a microfluidic PCR reactor, a passive diffusional micromixer reactor, and a microfluidic LDR reactor was separately designed and fabricated. Fluidic interconnects were also developed to make a fluidic passage between the functional components. Polycarbonate substrates were micro-molded, using hot embossing with micro-milled brass mold inserts to make all microfluidic components. Developed microassembly using passive alignment features, fabricated on all components, was used to assemble the functional components with the fluidic interconnects using an adhesive bonding technique. Thermal simulations were employed to ensure uniform thermal distributions in the microfluidic PCR and LDR reactors, to isolate the mixing junction in order to avoid heat–induced bubble formation in the passive micromixer reactor, and to have minimal thermal crosstalk due to the asymmetric thermal zones in the PCR and the LDR reactors. A control system was developed to control temperatures enabling thermal cycling in the microfluidic PCR and LDR reactor. LDR products were produced using the module within an hour with DNA sample, which had the ratio of 1:200. Total reaction time was about 67 minutes. By applying an enzyme as a purification of PCR products, a LDR analysis can be optimized and minimized to reduce the false positive signals and inconstant results generated by PCR products during the LDR. The purification system allowed us to successfully quantify the amount of mutant alleles in the genomic DNA. The high degree of accuracy in this module can also facilitate the detection of low-frequency point mutation occurred in other functional genes. This module, fabricated using replication technologies of polymers will be able to supply low cost, disposable detection tools for known disease-causing mutations and also expand to other PCR-based detection assays in diagnostic applications.
Document Availability at the Time of Submission
Release the entire work immediately for access worldwide.
Lee, Tae Yoon, "A multi-function, disposable, microfluidic module for mutation detection" (2009). LSU Doctoral Dissertations. 2120.
Michael C. Murphy