Effect of DNA extraction technique on theinfluences genetic detection of goldfish in an aquatic environment
John DeBuysser
Marian High School
1311 South Logan
Mishawaka, Indiana 46544
ACKNOWLEDGEMENTS
I thank the David Lodge lab located in Galvin Hall at the University of Notre Dame for providing me with the space and equipment necessary to complete my research. Also, I would like to thank the Indiana Junior Academy of Sciences for providing me with a research grant to fund part of the research. Thanks are also due to Matthew Barnes, my mentor at the lab who provided invaluable insight in the conception and completion of this project, and Mr. Andrzejewski, my sponsor and teacher of the Advanced Research class at Marian High School.
TABLE OF CONTENTS
List of Tables……………………………………………………………………….4
List of Figures
Introduction…………………………………………………………………………5
Materials and Methods……………….……………………………………………..8
Results……………………………………………………………..……………….11
Discussions and Conclusions……………………………………………………....11
References……………………………………………………………………….....12
LIST OF TABLES
TABLE 1 DNA Extraction Treatments 10
INTRODUCTION
Biological invasions are a problem for the indigenous communities or organisms. As invasive species become established outside their normal range, more pressure is placed on existing ecosystems for resources. Often the invasive species replaces the native species in the environment, as the invasive species compete more aggressively for resources. This is seen in the spread of the American bullfrog to many countries, including France, where its prolific nature and insatiable appetite quickly outcompete and remove native species (Ficetola, 2008). The same mode of operation can be seen in the spread of Asian carp species in the US in the Mississippi Valley region, where their voracious appetites and large adult sizes have led to the decline in biodiversity of the affected areas due to competition for resources (Jerde, 2011). Biological invasions are significant problem for the environment and must not be overlooked.
Invasives also have significant effects human society. Zebra mussels, which are now commonly found throughout the Great Lakes and other freshwater systems, are a species which is not native to the area and can be economically costly due to their propensity to clog pipes. Also, invasive aquatic plants, such as Eurasian watermilfoil, can interfere with recreation such as boating, swimming, and fishing by its prolific nature and tendency to get caught in boat propellars. Diseases and parasites are also a noteworthy concern when talking about invasives, such as the presence of feral swine and their propensity to pass diseases to livestock. Invasive species negatively affect society and can be costly to deal with.Biological invasions are significant problem for the environment and must not be overlooked.
There is much incentive to Controlling or prevent or controling biological invasions, as are of vital importance. iInvasive species are often known for their aggressive or destructive tendencies which can be costly to repair. Invasive species can be difficult, if not impossible, to completely eradicate once established and will invariably lead to more money being spent. Thus, proactive measures of prevention and/or rapid response to unnatural introductions are very important, and., and every effort is necessary to prevent their spread to other area. Due to the difficulty of complete eradication of a species once established, proactive measures are more effective, but there are ways to try to control established invasive species. However, it must be stressed that significant monetary outlayw can be avoided by preventing the spread of the species in the first place. Any tools that would help with early detection of invasive species should be developed.Control and prevention of spread of invasive species is of utmost importance.
Environmental DNA (eDNA) testing surveillance is a tool which is very useful for ecological surveys (Goldberg, 2011). It is a process which involves looking for genetic evidence, such as cells and DNA contained in materials such blood, feces, and hair, of the target organism in the environment, much in the same way that evidence at a crime scene is analyzed to prove that a suspect was there. To manage a species, invasive or not, a full knowledge of the population size, dispersal, and other factors are required. This is where environmental DNA testing is extremely beneficial, as it provides accurate data on a population by examining the presence of a target species with less effort compared to that of some traditional surveying methods (Jerde, 2011). Also, it is an effective instrument for working with secretive species in which it would be rare to physically spot the organism, as in the case of Asian carp species threatening to invade the Great Lakes (Jerde, 2011). Environmental DNA testing is an important asset in identifying and controlling biological invasions.
Calibration experiments are necessary to improve the accuracy and understanding of the results of DNA testing. By providing a controlled setting in which the number of unknown variables can be reduced, important findings can be realized such as the sensitivity of DNA techniques, how long DNA from an aquatic organism will persist in the environment (Dejean, 2011), or even how the results could eventually be used to determine population size. Calibration studies help to increase the effectiveness of DNA testing.
The research conducted is a series of two calibration studies. The first study focused on the persistance of goldfish DNA in an aquatic environment, however this research was met with the issue of not getting during the eDNA extraction process. With this in mind we refined our question and began a new study A previous research project involved testing the effects of humic acids on the detection and persistence of goldfish DNA in an aquatic environment. However, this research was unexpectedly cut short when an unknown issue with DNA extraction prevented completion of the project. The issues met in the in the experiment have provided the basis for which this new study will focus on, which is comparing the efficiencies of different DNA extraction methods. We isolated three main variables we are most interested in examining to see their effects on the extraction process. Three main comparisons that will be made revolve around whether filtering or precipitation should be used during DNA extraction, if Protinase K should be used in certain sample conditions, and whether there is a difference in performance between chemical supplied by a commercial kit or those that are custom mixed. The goal is that this study will clear up the issues encountered initially with eDNA extractionn, and thus allow the completion of the original project.
Filtration or precipitation during DNA extraction have both been proven to be highly successful. In a study on the population of bullfrogs in ponds in France, the precipitation method was used during DNA extraction with great success (Ficetola, 2008). In another study focusing on the spread of Asian carp is river and canal systems, the filtration method was used during DNA extraction and also yielded good results. This research project will employ both of these methods to determine if there are significant differences in their performance.
Protinase K is an enzyme which breaks down proteins and is often used in DNA extraction procedures to remove miscellaneous DNA from the sample. However, during an extraction an observation was made that the use of Protinase K resulted in a cloudy appearance of some samples. This posed the question ofremains whether this enzyme is necessary in controlled experiments where there is only small quantities miscellaneous DNA in the sample, and if in fact the enzyme does more harm than good in these situations by preventing DNA amplification and detection during PCR or qPCR procedures.
In a laboratory setting with ready access to a great supply of chemicals, it is often common for scientists to mix the chemicals themselves used in DNA extraction processes themselves to and cut costs by avoiding the expensive commercially available DNA extraction kits. However, is there a significant lost in performance of the extraction by usingdifference in the efficiency between custom mixed chemicals? This is a question we are looking into to see if or thosethere are any differences in yields between the two options, and if the differences would necessitate the use of the commercial kit over the mixed chemicals. purchased in a kit?
MATERIALS AND METHODS
The first calibration study conducted involved testing the persistence of goldfish DNA in an aquatic environment. Research involving non-human vertebrates or human subjects was conducted under the supervision of an experienced teacher or researcher and followed state and federal regulatory guidance applicable to the human and ethical conduct of such research. A series of four 20 L buckets were filled with 12 L of tap water and allowed to equalize for 24 hours, after which one Carassius auratus (goldfish) specimen was transferred to each of the buckets except for one bucket which was the negative control. 250 mL water samples were collected from each bucket immediately before and after the goldfish were introduced. The goldfish were left in the buckets for 24 hours to introduce a source of eDNA into the buckets, after which time the goldfish were removed. 250 mL water samples were collected from the buckets after goldfish removal and then every 24 hours for two weeks. All water samples collected were immediately filtered through a Millipore 10.0 mm filter membrane using vacuum filtration methods and the filters were then stored in a freezer at -20°C until extractions could be completed.
eDNA extractions were completed using CTAB DNA extraction procedure and chemicals mixed in the lab. The process for CTAB DNA extraction of samples we used was to combine 700 mL CTAB buffer and 20 mL Protinase K in a 2 mL tube containing the filter membrane. The tube was vortex mixed and then put in an incubator at 55°C for 4 hours or overnight. Then 700 mL of chloroform : isoamylalcohol (24:1) was added, vortex mixed for 5 seconds, and then spun in a centrifuge at room temperature at 13000 RPM for 10 minutes. The supernatant was then transferred via pipette to a new tube, and 1 mL of isopropanol was added and then mixed via several inversions of the tube. The tube is then stored in a freezer at -20°C for a minimum of 4 hours. Next, the samples were removed from the freezer and spun for 20 minutes at room temperature at 13000 RPM in a centrifuge. 500 mL of 70% ethanol was added and then centrifuged for 10 minutes. The ethanol was poured out, another 500 mL of 70% ethanol was added and then centrifuged again. The ethanol was again poured out and then dry tubes were placed in an incubator for 5- 15 minutes at 37°C. After the sample dried, 50 mL of T.E. was added to suspend the extracted DNA and then the tube was stored in a refrigerator overnight.
Next, the samples were prepared for qPCR to detect and quantify the amount of goldfish DNA present. In qPCR the extracted DNA is combined with chemicals that target a specific DNA sequence common only to the target organism and then cycled through a series of temperature gradients. During this process the DNA contained in the sample is denatured, or ‘unzipped’, and then replicated if it matches the matches the specific DNA segment coded for by the chemicals. This way, only the target organism’s DNA is amplified, while all the other miscellaneous DNA in the sample is not. To quantify the amount of DNA in each sample, a laser beam is shot into the sample many times through out the process and ‘counts’ the amount of DNA present. For this process 5 uL of the suspended DNA and 15 uL of a qPCR master mix were added to the wells of a qPCR plate. The master mix was composed of 10 uL Power SYBR green PCR master mix, 0.6 uL goldfish-specific forward primer, 0.6 uL goldfish specific reverse primer, and 3.8 mL H2O per reaction. The reaction began with a 10 minute step at 95° to activate the DNA polymerase enzyme contained within the master mix followed by 40 cycles of 94° for 15 seconds, 53°C for 15 seconds, and 72°C for 30 seconds.
The next study was then begun on testing the influences of DNA extraction technique on the genetic detection of goldfish in an aquatic environment. Three main comparisons made in this studythat will be made revolve around whether filtering or precipitation should be used during DNA extraction, if Protinase K should be used in certain sample conditions, and whether there is a difference in performance between chemical supplied by a commercial kit or those that are custom mixed. To complete this study, water samples were collected directly from the
A 75 L goldfish holdingglass aquarium set up in the lab solarium and subjected to rium with air stones for aeration and goldfish (Carassius auratus) is used as the source of aquatic environmental DNA. Water samples are collected from aquarium and subjected to one of the six treatment protocols as outlined in Table 1.
TABLE 1
DNA Extraction Treatments
Treatment / Collection Method / Extraction Method1 / Filter / CTAB+Protinase K
2 / Filter / CTAB-Protinase K
3 / Filter / MoBio Power Water DNA Isolation Kit
4 / Precipitation / CTAB+Protinase K
5 / Precipitation / CTAB-Protinase K
6 / Precipitation / Qiagen DNEasy kit
Treatment 1 used the exact same eDNA extraction procedures as outlined in the first study. The only difference between treatments 1 and 2 was that Protinase K was not used during extraction. Treatment 3 still used filtration as the method of eDNA collection, but used the commercially available MoBio Power Water DNA Isolation Kit and its directions during the extraction. Treatments 4, 5, and 6 used precipitation as the method of DNA collection. In the filtration process, a vacuum filtration station is used to filter the collected water samples through a 10.0 mm Millipore filter membrane. This membrane is then removed and stored in a freezer at -20°C until ready to begin the process of DNA extraction. In the precipitation process, 1.5 mL of sodium acetate and 35 mL of ethanol wereare added to a 15 mL water of sample water collected from the goldfish holding aquarium and then centrifuged at 5500g for 35 minutes to form a pellet, which wasis then ready for the next step of extraction. Treatment 4 used CTAB extraction with Protinase K, whereas treatment 5 did not use Protinase K during the CTAB extraction. The extraction for treatment 6 was completed using the commercially available Qiagen DNEasy kit. After all the extractions were completed the samples were prepared for qPCR and run though qPCR using the same procedures used in the first study.to begin the process of DNA extraction