Novel Methods of Phage DNA Separation in a Mixed Sample & Challenges of Gel Electrophoresis
Department
Biological Sciences
Faculty Advisor
Mauricio Rodriguez-Lanetty
Start Date
30-9-2020 1:00 PM
End Date
30-9-2020 2:00 PM
Abstract
Bacteriophages (or phages) are one of the most abundant organisms on the planet, having an estimated population of 1 x 10 These viruses not only have a large presence in many environments, but also have a high degree of genetic diversity due to horizontal gene transfer, exchanging genes between themselves and sometimes even their bacterial hosts to quickly adapt to their surroundings. This unique characteristic allows them to better circumvent bacterial defenses and successfully infect their host; phages are organisms of interest to researchers that wish to create novel gene therapies to treat bacterial illnesses and to fight against antibiotic resistance for this reason. Despite such ground-breaking applications, not many phages have been extensively studied in this field due to their vast population, meaning that there are still many more that have yet to be discovered. With this, then scientists require more efficient and precise tools to more readily be able to identify different phages in field samples in order to fill this knowledge gap. As a result, this study aims to explore (1) the limitations of different methods of separating phage DNA in a mixed DNA sample, and (2) utilizing one of these techniques to identify possible novel phage DNA sequences in a sample collected for the SEA-PHAGES program in 2018. Several established methods exist for separating DNA and identifying novel sequences, such as gel electrophoresis paired with Southern blotting, though newer technologies are working on the micro- and even nano-scale (e.g. "lab-on-a-chip" devices on Plexiglass substrates, sieving matrices for the nanoscale, etc.). Each of these methods have their benefits (e.g. increased precision in separating different DNA sequences) but they concur certain limitations (e.g. limited access to equipment/materials to build separation apparatus); even though the newer methods of DNA separation are not utilizing electrophoresis principles, gel electrophoresis is still the most widely utilized DNA separation technique in the laboratory. It is for this reason that gel electrophoresis was the chosen method for identifying possible novel phage DNA sequences in the 2018 SEA-PHAGES sample, where the following restriction enzymes were utilized: HindIII, HaeIII, ClaI, EcoRI-HF, BamHI-HF; the protocol was obtained from the Phagehunting Program within SEA-PHAGES. Preliminary gels showed some DNA bands running in the opposite direction of the electrical current (moving up to the negative charge instead of down to the positive); this was consistent across all the restriction digests. Future research is to be conducted on why this could be occurring, as well as other challenges that may be encountered when performing gel electrophoresis specifically with phage DNA.
File Type
Event
Novel Methods of Phage DNA Separation in a Mixed Sample & Challenges of Gel Electrophoresis
Bacteriophages (or phages) are one of the most abundant organisms on the planet, having an estimated population of 1 x 10 These viruses not only have a large presence in many environments, but also have a high degree of genetic diversity due to horizontal gene transfer, exchanging genes between themselves and sometimes even their bacterial hosts to quickly adapt to their surroundings. This unique characteristic allows them to better circumvent bacterial defenses and successfully infect their host; phages are organisms of interest to researchers that wish to create novel gene therapies to treat bacterial illnesses and to fight against antibiotic resistance for this reason. Despite such ground-breaking applications, not many phages have been extensively studied in this field due to their vast population, meaning that there are still many more that have yet to be discovered. With this, then scientists require more efficient and precise tools to more readily be able to identify different phages in field samples in order to fill this knowledge gap. As a result, this study aims to explore (1) the limitations of different methods of separating phage DNA in a mixed DNA sample, and (2) utilizing one of these techniques to identify possible novel phage DNA sequences in a sample collected for the SEA-PHAGES program in 2018. Several established methods exist for separating DNA and identifying novel sequences, such as gel electrophoresis paired with Southern blotting, though newer technologies are working on the micro- and even nano-scale (e.g. "lab-on-a-chip" devices on Plexiglass substrates, sieving matrices for the nanoscale, etc.). Each of these methods have their benefits (e.g. increased precision in separating different DNA sequences) but they concur certain limitations (e.g. limited access to equipment/materials to build separation apparatus); even though the newer methods of DNA separation are not utilizing electrophoresis principles, gel electrophoresis is still the most widely utilized DNA separation technique in the laboratory. It is for this reason that gel electrophoresis was the chosen method for identifying possible novel phage DNA sequences in the 2018 SEA-PHAGES sample, where the following restriction enzymes were utilized: HindIII, HaeIII, ClaI, EcoRI-HF, BamHI-HF; the protocol was obtained from the Phagehunting Program within SEA-PHAGES. Preliminary gels showed some DNA bands running in the opposite direction of the electrical current (moving up to the negative charge instead of down to the positive); this was consistent across all the restriction digests. Future research is to be conducted on why this could be occurring, as well as other challenges that may be encountered when performing gel electrophoresis specifically with phage DNA.