Chris Sheldon, Ryan Dawson Section 403 11/27/13 Genetic Sequencing of Micropogonias undulatus Abstract Over the course

of five weeks DNA was extracted from a sample, amplified with Polymerase Chain Reaction (PCR), visualized with gel electrophoresis, put through a genetic sequencer, and finally analyzed with an online database. Tissue samples were cut from an organism and then DNA was extracted using cellular lysis and chemicals. PCR was run for 35 three and a half minute loops with an initial denaturation of 2 minutes and a final elongation of 10 minutes. Gel electrophoresis was run with a 1% agarose gel at 115 volts for 40 minutes. The gel electrophoresis showed that PCR worked, however there was not enough sample DNA to fully show results. However, a sequence was given when the sample DNA was put through the genetic sequencer. The data from the sequencer was entered into a bioinformatics program called BLAST. BLAST analyzed and compared the sequence with sequences from other closely related species. The most common sequence match was from a study done by McCusker showing that our genetic sequence matched Micropogonias undulatus. A phylogenetic tree was constructed from the BLAST program with all related species, showing that the sample was most closely related to M. undulatus and Roncador stearnsii. Using the data from both McCuskers study and the phylogenetic tree, it was concluded that the sample if from M. undulatus. However, since gel electrophoresis did not show any results for the sample, more research is needed to confidently identify the sample.

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Chris Sheldon, Ryan Dawson Section 403 11/27/13 Introduction

The purpose of this project is to, over the course of five weeks, collect genetic information from an environmental sample (a fish). The reason this took five weeks is because there were five different sections to it: Nucleic Acid Extraction, Polymerase Chain Reaction, Gel Electrophoresis, Genetic Sequencing, and Bioinformatics. Each of these sections was the topic of one lab day. The environmental samples were supplied for us the day of the nucleic acid extraction and we chose to use the Atlantic Croaker, Micropogonias undulatus. Micropogonias undulatus is of the Sciaenidae family. They can be found in the western Atlantic from Massachusetts to northern Mexico down to southern Brazil and Argentina. M. undulatus usually range in length from 18-30 cm and can live up to 5 years. Adults can usually be found over mud and sandy mud bottoms in estuaries and coastal waters. Nursery and feeding grounds are usually found in these same waters as well. The diet of M. undulatus consists mainly of worms, crustaceans, and smaller fishes(H. Dickson Hoese 1998). The purpose of the Nucleic Acid Extraction is to isolate DNA from tissue samples of our selected fish. In order to isolate this nucleic acid, there are three requirements that must take place: the cell structure and membranes have to be disrupted, any DNA degrading substances have to be inactive, and finally, the nucleic acids have to be separated from any substrate matrix and cell components. There are three different techniques that can be used to break down the cell structures. A common technique is a freeze/thaw cycle. Rapidly freezing, which causes internal liquids in the cell to expand, and then rapidly thawing over and over puts stress on the cellular membrane and could cause it to break. An advantage of this technique is that freezing will inhibit enzymes responsible for breaking down nucleic acids. Another way to break down the cell membrane is a process called sonification. This involves shooting sound waves at the sample which will agitate particles and breaks down cell membranes. However, care must be taken; sonification can also disrupt DNA structure. The next step for nucleic acid extraction is to deactivate DNA degrading which can be done with

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chemicals. Some common examples of chemicals that can be used are Guanidinium

Chris Sheldon, Ryan Dawson Section 403 11/27/13

thiocyanate and 2-mercaptoethanol. The last step is to isolate the DNA from any other cell structures. This can be done by adding isopropanol or Ethanol which causes the DNA to precipitate out of the solution. The DNA will precipitate out because the polarity of these alcohols is much lower than that of water, which is what the DNA is originally soluble in. The next section is to do a Polymerase Chain Reaction (PCR) in order to multiply the small amount of DNA we extracted. PCR has three steps to it: denaturation, annealing, and elongation. Each step has a certain time limit and temperature its supposed to be run at, and all the steps are repeated multiple times. The first step, denaturation, is the separation of DNA by increasing the temperature of the reaction. The increase of temperature disrupts the hydrogen bonds between base pairs. The temperature is decreased for the second step, the annealing step. The temperature the reaction is then set to depends on the type of primer being used; this allows the primer to attach to the single stranded DNA. The third step raises the temperature again and is called the elongation step. The temperature is raised again in order to maximize the efficiency of the enzyme in the reaction. This is the step when the polymerase makes complimentary DNA to the template strand. The combination of DNA extraction and PCR amplification is a technique used across multiple taxa, such as a study done by Cubero, where DNA was extracted and amplified from fungi (Oscar F. Cubero 1997). After PCR the next step is gel electrophoresis. Nucleic acids have a negative charge, so an electrical current and frictional resistance (in the form of an agarose gel) can be used to separate the nucleic acid fragments. There are several factors that affect how fast and far the nucleic acid fragments can travel in the gel. The higher the voltage applied the faster the fragments will travel. The percent of the agarose in the gel also determines the size of the fragments that will efficiently separate, with higher percentages of agarose allowing from longer fragments to be separated.

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The second to last step is to sequence the duplicated DNA so that the species can be determined. There are many different kinds of sequencing techniques; the kind we use in

Chris Sheldon, Ryan Dawson Section 403 11/27/13

class is called the Sanger sequencing method. In the Sanger method, the nucleotide bases will glow a certain color depending on which base it is. The fragments are separated with electrophoresis and then passed by a laser. The laser detects the specific color and the software identifies which nucleotide it represents. There are two other types of sequencing techniques, Illumina sequencing and 454 Pyrosequencing. Illumina sequencing can sequence and generate large amounts of DNA but has a higher error rate of 1.5%. The 454 pyrosequencing can do 400,000 reads per run but can only recognize fragments between 200-300 base pairs. After the data has been sequenced the results from the sequencer have to be examined with software, this step is called bioinformatics. The National Center for Biotechnology Information has an online database that can be used to examine sequencer information. The program used to examine the info is the Basic Local Alignment Search Tool, or BLAST program. The BLAST program runs the genetic sequence through its database and shows matches if finds to any of the species. Using this information the species of the sequenced DNA can be identified.

Methods DNA Extraction Tissue samples were taken from the muscle tissue of M. undulatus weighing 100mg and placed in a micro centrifuge tube. A tube with no sample in it will be used as a negative control to make sure that there is no contaminant in any of the solutions. Gloves were worn during the entire experiment in order to ensure no contamination.

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Chris Sheldon, Ryan Dawson Section 403 11/27/13 A Nuclei Lysis Solution was added to each tube (600uL) and allowed to homogenize for 10 seconds. Three microliters of the RNase solution was added to each, mixed, and incubated for 15 minutes at 37 degrees C. After this, the solution was cooled to room temperature and 200uL of Protein Precipitation solution were added to both tubes. This mixture was vortexed, chilled on ice for five minutes, and centrifuged at 13000-16000 for four minutes. The supernatant was then added to a fresh tube that contained 600uL of isopropanol and mixed by inversion. This was again centrifuged at 13000-16000 for one minute. The supernatant was removed without disturbing the pellet and 600uL of 70% ethanol was added. This was mixed, centrifuged at 13000-16000 for another minute, and the ethanol was allowed to evaporate. The DNA was then rehydrated with 100uL with DNA rehydration solution in an incubator for one hour at 65%. The two tubes were then left with the TA for storage until the following week. PCR (Polymerase Chain Reaction) For the PCR part of this experiment we added a positive control to make sure that PCR worked. 35uL of Polymerase Master Mix was added to each tube and 5uL of the DNA sample was added to the corresponding tube. Total volume for each tube was 40uL. Each tube was flicked and tapped on the table in order to ensure the solution was completely mixed. The TA then used a thermal cycler to perform the denaturation, annealing, and elongation process of the DNA fragments. The program on the thermal cycler first ran an initial denaturation for 2 minutes at 94C. Then it performed a continuous loop of Denaturation for 30 seconds at 94C, Annealing for 2

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Chris Sheldon, Ryan Dawson Section 403 11/27/13 minutes at 55C, and Elongation for 1 minute at 72, 35 times. After this the thermal cycler performed the final elongation for 10 minutes at 72C. It was then held at 4C until Gel electrophoresis could be performed. Gel Electrophoresis (Making Agarose Gel) To make an agarose gel, 0.5g of Molecular Grade agarose was added to 50mls of 1X TAE buffer. It was then microwaved for 30 seconds and stirred until all agarose had dissolved. Once mixture had cooled 3 !L of Ethidium Bromide was added and mixed. After this, it was poured directly into the gel tray to cool and solidify for 25 minutes. Visualizing the PCR On a piece of parafilm three, 2.5 !L dots of 6X loading dye were placed. Then 2.5 !L of each PCR sample was added to a dot of loading dye. Each dot was mixed using a pipette then placed into the gel and gel electrophoresis was run at 115 V for 40 minutes. A Gel Imaging system was used to photograph the gel. Genetic Sequencing and Bioinformatics For each PCR tube containing 1!L, 9!L Master Mix; comprised of 2!L Sequencing buffer, 0.5!L BigDye Terminator Mix, 1!L Forward Primer, and 5.5!L of water, was added. This was then put into the thermal cycler and cycled for 45 minutes at 95C for 1 minute, 15 cycles of 95C for 0.10 seconds, 50C for 0.05 seconds, 60C for 1.15 minutes, 5 cycles of 95C for: 10 seconds, 50C for 0:10 seconds, and 60C for 1:30 minutes, and 5 cycles of 95C for 0:10 seconds, 50C for 0:05 seconds, and 60C for 2:00 minutes. After the cycling the unknown DNA sample was cleaned with 27.5!L of BDX terminator. The tubes were placed into a vortex

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Chris Sheldon, Ryan Dawson Section 403 11/27/13 shaker for 30 minutes at 2000 RPM and then centrifuged for one minute at 2000 RPM. The supernatants were then pipetted from the PCR tubes into the sequencing tube plate. The sequencing was then performed using an ABI Prism 310 Genetic Analyzer. The sequence was then matched and compared using a blast program on the NCBI website. Using the NCBI site, a phylogenetic tree was made to show evolutionary relationships. Results The gel electrophoresis determined that there was DNA in the positive control (C+) well. However, no results were seen for our negative control (C-) and sample (S) lanes (Figure 1). Despite the fact that the electrophoresis showed no results for the sample, the genetic sequencing was able to detect enough DNA in our sample to determine the genetic sequence of the COI gene. When the genetic sequencer information was entered into the BLAST program, the closest gene match given was that of the Micropogonias undulatus (Figure 2). The phylogenetic tree showed that the unknown sample was most closely related to Micropogonias undulatus and Roncador stearnsii (Figure 3).
S C+ C-

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Chris Sheldon, Ryan Dawson Section 403 11/27/13

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Figure 2. BLAST results of sample sequence for the COI gene

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Chris Sheldon, Ryan Dawson Section 403 11/27/13

" Figure 3. Phylogenetic tree of the COI gene relation Discussion The fact that the gel electrophoresis showed a faint result in the positive control lane shows that PCR worked, but not very efficiently. Primer dimers could have showed up earlier than expected and inhibited the reaction from successfully completing. There are two possible explanations for why the sample lane showed nothing even though PCR was successful: either extraction did not work or there was not enough of the sample DNA for the electrophoresis to run properly. However, since the genetic sequencer was able to determine the sequence of the COI gene from the sample we can conclude that the latter of these is the probable factor.

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Chris Sheldon, Ryan Dawson Section 403 11/27/13 The COI from the sample was compared to the COI gene in the BLAST program. This showed a 98% similarity in the gene between our data and the data of a study done by McCusker (2012). From this we are able to determine that our sample DNA could be from the fish Micropogonias undulatus. Also, noted in McCuskers study was that the similarity in populations within this species showed a 98% similarity, which explains the difference of our sample and McCuskers (McCusker MR 2013). The phylogenetic tree made by the BLAST program also determined that our sample was closely related to M. undulatus and Roncador stearnsii. Using the data from both the phylogenetic tree and McCuskers study, we concluded that the sample DNA was from M. undulatus. Since the gel electrophoresis did not successfully show any results for the sample DNA more research needs to be done on this same sample to solidify this conclusion. References
H. Dickson Hoese, R. H. M. ( 1998). Fishes of the Gulf of Mexico. College Station, Texas A&M University Press. McCusker MR, D. D., Van Guelpen L, Kenchington E, Bentzen P. (2013). "Barcoding Atlantic Canada's commonly encountered marine fishes." Molecular Ecology Resources 2: 177-188. Oscar F. Cubero, A. C., Jamshid Fatehi, Paul D. Bridge (1997). "DNA extraction and PCR amplification method suitable for fresh, herbarium-stored, lichenized, and other fungi." Plant Systematics and Evolution 216: 243-249.

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