2025 Summer Undergraduate Research Program (SURP) symposium
Location
Dr. Ken Budke Family Auditorium, Schindler Education Center, University of Nothern Iowa
Presentation Type
Open Access Poster Presentation
Document Type
poster
Abstract
Caves offer a unique environment to study microbial life that survives under extreme nutrient limitation. To this extent, 16s metabarcoding serves as a sort of “DNA fingerprint” that allows us to identify and study the microbial composition of these environments, but there are hurdles that must be overcome to obtain enough DNA for effective and reliable 16s sequencing. It is important to maximize the amount of DNA that can be extracted from the Wind Cave samples because it is an extremely low biomass environment and sufficient DNA for sequencing can be hard to get. Wind Cave National Park serves as a relatively accessible cave microbiome to study. It is a limestone karst cave, meaning it is largely made up of the mineral calcite, which can inhibit DNA extraction because of positive charges in the mineral structure trapping negatively charged DNA on its surface. Our lab has previously worked on developing a DNA extraction protocol suited for high DNA yield from environmental samples, including many experiments with Wind Cave samples, yet we still obtain low yields. I’ve been developing a “mineral DNA extraction method” based on the principle of repeated freeze-thaw, chemical, and enzymatic lysis with the addition of pyrophosphate to act as a blocking agent and release trapped DNA. To test this new DNA extraction method, Wind cave samples from different locations were supplemented with E.coli and extracted two sets of samples in parallel using the “lab DNA extraction protocol” and the “mineral DNA extraction protocol”. Utilizing the “mineral DNA extraction” obtained a significantly higher yield of DNA compared to our previous lab protocol and enables robust downstream DNA analysis with a more complete sample.
Key words: cave microbiology, DNA extraction, mineral abundant DNA samples Publish
Start Date
1-8-2025 11:00 AM
End Date
1-8-2025 1:30 PM
Event Host
Summer Undergraduate Research Program, University of Northern Iowa
Faculty Advisor
Marek Sliwinski
Department
Department of Biology
Copyright
©2025 Treytun Garcia and Marek Sliwinski
File Format
application/pdf
Recommended Citation
Garcia, Treytun and Sliwinski, Marek, "Developing a DNA Extraction Method for Mineral Abundant Limestone Cave Samples" (2025). Summer Undergraduate Research Program (SURP) Symposium. 28.
https://scholarworks.uni.edu/surp/2025/all/28
Developing a DNA Extraction Method for Mineral Abundant Limestone Cave Samples
Dr. Ken Budke Family Auditorium, Schindler Education Center, University of Nothern Iowa
Caves offer a unique environment to study microbial life that survives under extreme nutrient limitation. To this extent, 16s metabarcoding serves as a sort of “DNA fingerprint” that allows us to identify and study the microbial composition of these environments, but there are hurdles that must be overcome to obtain enough DNA for effective and reliable 16s sequencing. It is important to maximize the amount of DNA that can be extracted from the Wind Cave samples because it is an extremely low biomass environment and sufficient DNA for sequencing can be hard to get. Wind Cave National Park serves as a relatively accessible cave microbiome to study. It is a limestone karst cave, meaning it is largely made up of the mineral calcite, which can inhibit DNA extraction because of positive charges in the mineral structure trapping negatively charged DNA on its surface. Our lab has previously worked on developing a DNA extraction protocol suited for high DNA yield from environmental samples, including many experiments with Wind Cave samples, yet we still obtain low yields. I’ve been developing a “mineral DNA extraction method” based on the principle of repeated freeze-thaw, chemical, and enzymatic lysis with the addition of pyrophosphate to act as a blocking agent and release trapped DNA. To test this new DNA extraction method, Wind cave samples from different locations were supplemented with E.coli and extracted two sets of samples in parallel using the “lab DNA extraction protocol” and the “mineral DNA extraction protocol”. Utilizing the “mineral DNA extraction” obtained a significantly higher yield of DNA compared to our previous lab protocol and enables robust downstream DNA analysis with a more complete sample.
Key words: cave microbiology, DNA extraction, mineral abundant DNA samples Publish
