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DNA Denaturation Mapping in Nanochannels for Bacterial Identification

Scaramuzza, Stefano LU (2015) FYSM60 20142
Department of Physics
Solid State Physics
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... (More)
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. (Less)
Popular Abstract
The DNA (Deoxyribonucleic acid) is a molecule found in every living organism and bears information which is unique for its carrier. Reading this information would thus allow to identify any kind of organism. The existing methods to read the content of the DNA are most of the times either slow or expensive. In this project, a novel technique to visualize the information stored in the DNA is explored. This technique has the potential to simplify and speed up the reading process, making it suitable for the identification of bacteria and thus making it a powerful diagnostic tool.
In this technique a DNA molecule is inserted into tiny channels (nanochannels) that force the molecule to stretch out. The DNA is then labelled with a special dye... (More)
The DNA (Deoxyribonucleic acid) is a molecule found in every living organism and bears information which is unique for its carrier. Reading this information would thus allow to identify any kind of organism. The existing methods to read the content of the DNA are most of the times either slow or expensive. In this project, a novel technique to visualize the information stored in the DNA is explored. This technique has the potential to simplify and speed up the reading process, making it suitable for the identification of bacteria and thus making it a powerful diagnostic tool.
In this technique a DNA molecule is inserted into tiny channels (nanochannels) that force the molecule to stretch out. The DNA is then labelled with a special dye that falls off at specific regions of the DNA upon heating. These regions depend on the information of the DNA. Under the microscope, only regions containing the dye are visible. This pattern of bright and dark regions along the DNA can be interpreted as a barcode, which is unique for every organism. By comparing this barcode with a database of theoretically generated barcodes, the DNA and its carrier can be identified.
In this project, fundamental studies of the technique have been performed and an attempt at identifying bacteria was made. It could be shown that it takes 10 minutes for the barcode to appear on the DNA when heated. It was also observed that the temperature at which the barcode appears depends on the amount of dye and the level of confinement of the DNA. Further, a good agreement between experimental barcodes and theoretical barcodes could be demonstrated. Even though a large amount of bacterial DNA was analysed, an identification of the bacteria could not be realized yet. Improvements in the experimental method as well as in the data analysis are believed to improve the results. (Less)
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author
Scaramuzza, Stefano LU
supervisor
organization
course
FYSM60 20142
year
type
H2 - Master's Degree (Two Years)
subject
keywords
DNA denaturation mapping, bacterial identification, nanofluidics, microfluidics, single molecule imaging
language
English
id
4940948
date added to LUP
2015-02-10 21:04:24
date last changed
2015-02-10 21:04:24
@misc{4940948,
  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.},
  author       = {Scaramuzza, Stefano},
  keyword      = {DNA denaturation mapping,bacterial identification,nanofluidics,microfluidics,single molecule imaging},
  language     = {eng},
  note         = {Student Paper},
  title        = {DNA Denaturation Mapping in Nanochannels for Bacterial Identification},
  year         = {2015},
}