DNA Denaturation Mapping in Nanochannels for Bacterial Identification
Abstract: In the current work, partial denaturation mapping of DNA in nanochannels is studied with the goal to use this technique as a tool for the identification of bacteria. This method has the potential to simplify and speed up the diagnosis of bacterial infections. A partially denatured and YOYO-1-labeled DNA molecule displays a sequence specific pattern of fluorescent and non-fluorescent regions along the molecule. This pattern can be interpreted as a barcode. By stretching the DNA molecule inside nanofluidic channels, this barcode can easily be imaged with an optical microscope and compared with a database consisting of theoretically generated barcodes. The DNA is identified by finding the theoretical barcode that matches best to the experimental barcode. In this project, first fundamental studies of the denaturation pattern formation have been performed. Pattern formation has been studied as a function of time, confinement and fluorescent dye concentration. Second, experimentally acquired barcodes were compared to their corresponding theoretical barcodes to demonstrate the agreement between experiment and theory. Finally, identification of the strain specific DNA of S. pneumoniae, was attempted. The time it takes for denaturation patterns to form upon heating the DNA has been observed to be approximately 10 minutes. The difference in melting temperature between nano- and microconfinement was found to be in the range of 5-10 C. An effect of the fluorescent dye concentration on the melting temperature of DNA molecules was observed. Further, a good agreement between experimental and theoretical barcodes could be demonstrated. A large amount of denaturation maps of DNA fragments from different strains of S. pneumoniae were measured. However, successful identification of the bacterial DNA has not been realized yet. Potential ways to improve the results are discussed.
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