Doctor of Philosophy (PhD)
A number of genotyping methods have been developed for mutation analysis, each of which has its own unique advantage. DNA amplification via polymerase chain reaction (PCR) provides an unlimited supply of material for subsequent genetic analysis even in case where only a single copy of the DNA molecule is present in the sample. Research presented in this dissertation first examines the efficiency of in vitro amplification of single copy DNA with subsequent sequencing analysis of PCR product. The PCR products (amplicons) were investigated for possible alteration, distortions or mutations due to the amplification process. The sequencing data for single copy amplification indicated a read length of 424 bases could be achieved with read accuracy of 99.3%. A confocal detection system was constructed to monitor the fluorescence signature from single DNA molecule labeled with near infrared (NIR) dyes. The performance of the system was first tested by applying single photon burst technique to detect double-stranded DNA molecules in polymeric devices. Sampling efficiencies were investigated by physically narrowing the channel sizes of the microdevices and by applying electrokinetic focusing. The narrow channels showed 4 times improvement in a 15 µm channel compared to a 50 µm channel. Similar results were demonstrated in the focusing studies. Single molecule sizing of ë-DNA, M13, pUC19 and pBR322 DNA using single photon burst was also demonstrated. In addition single molecule detection of a NIR chromophore NN383 was analyzed in PC devices and a detection efficiency of 94% was achieved. The other application for the NIR system was in a new strategy for analyzing molecular signatures of disease states in real-time using single-pair fluorescence energy transfer (spFRET) coupled with ligase detection reaction (LDR) to rapidly detect single base mutations in codon 12 of K-ras oncogene which has high diagnostic value for colorectal cancer. LDR-spFRET provided the necessary specificity and sensitivity to detect single point mutations in as little 600 copies of human genomic DNA without PCR amplification at a level of 1 in 1000 wildtype sequences. In addition the assay demonstrated analysis times < 5 min.
Document Availability at the Time of Submission
Release the entire work immediately for access worldwide.
Wabuyele, Musundi Ben, "Genome analysis: mutation analysis using near infrared laser-induced fluorescence (NIR-LIF) and single molecule detection in microfluidic devices" (2003). LSU Doctoral Dissertations. 380.
Soper, Steven A.