Design of Plasmid Amplified DNA Building Block Synthesis System and Evaluation of Dendrimer-Like DNA Based Fluorescent Nanobarcodes
Methods for producing DNA building blocks with high purity and yield were investigated, including solid-phase DNA synthesis and plasmid amplified DNA synthesis (PADS). In addition, an analysis of the properties of dendrimer-like DNA (DL-DNA) as nanobiosensor was conducted to explore the viability of its real-world application. Four-armed dsDNA building blocks (X-DNA's) were successfully acquired using solid-phase synthesis. X-DNA consisted of 4 oligonucleotides that are partially complementary such that a cross-shaped dsDNA molecule is formed upon annealing. It was ligated to a 30bp dsDNA spacer immobilized onto micrometer-sized 6% cross-linked agarose beads via biotin-avidin interactions. A subsequent washing step was performed to rid the sample of non-X-DNA structures, and X-DNA was released from the spacer by restriction enzyme digestion. Gel electrophoresis of the product showed higher purity, 72% compared to 67.5% shown in the solution-hybridized X-DNA prior to solid-phase. Characterization of X-DNA was performed by ligation of 4 complementary hairpin loops which serve to close off all open dsDNA ends and prevent the structure from exonuclease digestion. Unchanged DNA concentration after 15 and 30 min of ExoIII digestion at 37oC was observed, confirming the synthesis of X-DNA. Plasmid amplified DNA synthesis takes advantage of the natural DNA producing system in Escherichia coli for high-yield production of plasmids containing sequence for three-armed DNA building blocks (Y-DNA). A nicking enzyme was used to produce a single-stranded break in the plasmid. ExoIII digestion at 37oC was performed to produce ssDNA plasmids. Annealing at 70oC causes a branched hairpin (Y-shape) to form on each ssDNA strand. Simultaneous digestion of the Y-shape hairpin by three enzymes produces Y-DNA. Single and combinational of enzyme digestion was applied to characterize the ssDNA plasmid, and determined to be a Y-shape structure. Lastly, fluorescent DNA nanobarcodes were analyzed for their purity, coding capability, compared to concentration-based coding method, as well as differential bleaching of green (G) and red (R) fluorescence. Pure populations of DNA nanobarcodes (4G1R, 2G1R, 1G1R, 1G2R, 1G4R) and multi-code mixtures, immobilized on 5.5um polystyrene beads, were obtained. The fluorescent intensities (R and G) were measured from 12-bit images taken by a wide-field microscope; the illumination source is a Mercury arc lamp and respective fluorescent colors obtained using green and far-red filters. The purity of each population was assessed by analyzing the magnitude of R/G fluorescent ratio standard deviation for each pure barcode populations (N>50beads). Comparison of the mean for each codes to a theoretical R/G ratio yield their codability. The DNA nanobarcodes were determined to be pure and their experimental R/G ratios conform to theoretical values, unlike concentration-based DNA barcodes. Bleaching analysis of red and green fluorodyes reveal that red dye bleach faster than green, however the ratio of R/G, and nanobarcodes, did not change significantly over time.
DNA; DNA Building Block; generic; nucleic acid engineering; barcode; Fluorescent barcode
dissertation or thesis