Gene-ID Using Simultaneous DNA Barcoding and Enzymatic Labeling

Abstract: Antibiotic resistance is an ever growing problem, and is considered one of the major challenges of modern medicine. In order to avoid further development and spread of multiresistance among bacteria, the use of antibiotics should be more restrictive, and more tailored to individual cases of infection. To facilitate more directed treatments, it is crucial to be able to quickly and reliably identify the strain of bacteria. A logical next step would be to directly be able to identify any genes coding for antibiotic resistance. To this end, I investigate the possibility of combining two methods of optical mapping of plasmids - DNA barcoding, and enzymatic labeling of specific genes. The end goal is to develop a single experiment in which both the plasmid type, and any genes coding for antibiotic resistance, can be identified. I focus mainly on three tasks - all related to the processing of experimental data. (1) Improving an earlier method for kymograph alignment, necessary for processing data from experiments using DNA in nanochannels. (2) Creating reproducible time averages of sparsely labeled barcodes, by attempting to exclude non emitting fluorophores. (3) Determining the number of fluorophores in a sparsely labeled barcode, by fitting a number of Gaussians to its reproducible time average. The results of all three tasks were promising, but as no data was available of plasmids labeled using both techniques of optical mapping, no definitive conclusions could be made about the approach.