Next Generation Sequencing of Putative Ranavirus Specific DNA Aptamers
Presentation Type
Poster
Faculty Advisor
Nina Goodey
Access Type
Event
Start Date
26-4-2024 11:15 AM
End Date
26-4-2024 12:15 PM
Description
Ranavirus has been detected in various amphibian species, leading to infections and ultimately mortality in frogs. It is crucial to be able to identify and track the virus in natural and commercial environments, but the current testing methods have major drawbacks because they are intricate, costly, and time-consuming. To address these challenges and potentially devise a fast field test delivering immediate results, we have used a laboratory technique known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to select single stranded DNA aptamers that bind to recombinant Frog virus 3 MCP (major capsid protein). Such an in vitro selection of single stranded DNA aptamers that bind this protein is thought to deliver high-affinity detection reagents that can be built into an easy and robust field test. Initially, a large pool of random DNA sequences is exposed to the FV 3 MCP protein. DNA sequences that bind to the MCP are retained, while non-binding sequences are washed away. The bound DNA sequences are then amplified through PCR (polymerase chain reaction) and subjected to further rounds of selection. Through multiple cycles of selection and amplification, the pool of DNA sequences becomes enriched for those that bind most tightly to the FV 3 MCP. The present work describes i) high throughput next generation sequencing efforts to determine putative MCP-binding single DNA aptamer sequences and ii) follow-up bioinformatics analysis to identify the best aptamer sequence candidates for subsequent testing in MCP binding assays. In preliminary results, we analyzed almost 300,000 sequences from an aptamer library that resulted from four SELEX rounds and over 400,000 sequences from a library that resulted from nine SELEX rounds. . Initial inspection of these sequences revealed the presence of some duplicate aptamer sequences and further bioinformatic analysis presented here will analyse these results in more detail... The presence of such duplicate aptamer sequences would suggest that enrichment took place during the SELEX process and that those sequences that occur multiple times in a library may be ones that bind to the major capsid protein target.
Next Generation Sequencing of Putative Ranavirus Specific DNA Aptamers
Ranavirus has been detected in various amphibian species, leading to infections and ultimately mortality in frogs. It is crucial to be able to identify and track the virus in natural and commercial environments, but the current testing methods have major drawbacks because they are intricate, costly, and time-consuming. To address these challenges and potentially devise a fast field test delivering immediate results, we have used a laboratory technique known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to select single stranded DNA aptamers that bind to recombinant Frog virus 3 MCP (major capsid protein). Such an in vitro selection of single stranded DNA aptamers that bind this protein is thought to deliver high-affinity detection reagents that can be built into an easy and robust field test. Initially, a large pool of random DNA sequences is exposed to the FV 3 MCP protein. DNA sequences that bind to the MCP are retained, while non-binding sequences are washed away. The bound DNA sequences are then amplified through PCR (polymerase chain reaction) and subjected to further rounds of selection. Through multiple cycles of selection and amplification, the pool of DNA sequences becomes enriched for those that bind most tightly to the FV 3 MCP. The present work describes i) high throughput next generation sequencing efforts to determine putative MCP-binding single DNA aptamer sequences and ii) follow-up bioinformatics analysis to identify the best aptamer sequence candidates for subsequent testing in MCP binding assays. In preliminary results, we analyzed almost 300,000 sequences from an aptamer library that resulted from four SELEX rounds and over 400,000 sequences from a library that resulted from nine SELEX rounds. . Initial inspection of these sequences revealed the presence of some duplicate aptamer sequences and further bioinformatic analysis presented here will analyse these results in more detail... The presence of such duplicate aptamer sequences would suggest that enrichment took place during the SELEX process and that those sequences that occur multiple times in a library may be ones that bind to the major capsid protein target.