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Sequencing, isolation and genomic analysis of brine shrimp (Artemia franciscana) genes Introduction The species Artemia franciscana holds a countless number of genes, all of which are dedicated to a specific purpose of function. The final goal of this experiment is to create a catalog of genes for the brine shrimp, along with their given role in the species, due to their overall usefulness in research projects. Messenger RNA and a process known as reverse transcriptase is used to create cDNA, which is then inserted into plasmid vectors, which are in turn placed in cells of E. coli. These bacterial cells are allowed to multiply via binary fission, creating a library of cDNA. We will then use this library to prepare, sequence and analyze the samples to cDNA to ultimately compare to other genes and discover the roles of brine shrimp. Other research projects have succeeded in isolating specific genes, which showed “Genomic clones coding for the Artemia franciscana sarco(endo)plasmic reticulum Ca-ATPase have been isolated.” (Ricardo Escalante & Leandro Sastre, 1993). Using the collaborative results from multiple students and schools, the projects hopes to find a relation between brine shrimp and other organisms and see which gene sequences are preserved over time. Materials Sequencing of Artemia was made possible with Miniprep Plasmid DNA, created and provided Rutgers University (Serial Number OAF 746.06) . The first sample we created for Gel Electrophoresis was a PvuII digest, which consisted of both the Miniprep DNA and the PvuII enzyme. We also created a control for our restriction digest , which was equivalent to the PvuII digest, without the actual enzyme. The purpose of this mixture was to have a base sample to verify if the enzyme is working properly. Our last digest was a PCR mix, which included a diluted version of our original DNA , and both SP and BP primers. We ran this mixture through a PCR machine, which repeatedly warmed and cooled the mixture and specific intervals, which allowed for a larger sample size that would be physically visible after Gel Electrophoresis. The length of the DNA sequences was shown using Agarose Gel, and power supply (with a combination of EtBr and 1x Buffer for a submergent liquid). First, the gel box was filled up with EtBr and 1x Buffer solution and agarose gel was placed inside (Figure 1). Then, pipette each solution (Figure 2) into a separate well (Figure 3). Then, hook up the gel box to a power supply (Figure 4) and run the power supply for 30 minutes. The results of the gel electrophoresis (Figure 5) show the 1 Kb DNA Ladder in well one, followed by the Control, PvuII, and PCR DNA, respectively. The lack of bands in both wells two and three suggest the sizes of the restriction digest inserts are very small. The visible DNA ladder for the PCR sample indicates the presence of an insert in the PCR. The size of this insert is estimated at 500 base pairs. Methods Results Figure 1: A correctly set up gel box, filled with agarose and submerged in a solution of EtBr and 1x Buffer Figure 3: Describes each well Well 1: 1 Kb DNA Ladder Well 2: Uncut, Control DNA Well 3: PvuII Digested DNA Well 4: PCR Plasmid DNA Figure 2: Shows micropipette and pipetting of different mixtures into wells Figure 4. Shows power supply, which connected to the gel box for electrophoresis. Well 1 Well 2 Well 3 Well 4 Figure 5. A picture of the agarose gel after electrophoresis, under a UV light. The different wells and their lengths are shown. Conclusion The results of the gel electrophoresis on the multiple DNA samples suggests the PCR worked and the presence of an insert at around 1200 base pairs. The next step would be to take this sequence and analyze it computationally through DNA visualization softwares such as 4Peaks or FinchTV. Also, running the DNA sequence through comparison databases such as BLASTn would be ideal, but due to time constraints, neither of these solutions are possible. There is also an issue with the accuracy of the gel electrophoresis, as due to human error, there may be an error during the creation or pipetting of the samples. 12,000 Base Pairs 5,000 BP 1,650 BP 850BP 200 BP Eric Lee References www.tinyurl.com/3zuu7p, for tutorials on the entire process. www.tinyurl.com/blastn, for BLASTn nucleotide comparison databases “Structure of Artemia franciscana Sacro/Endoplasmic Reticulum Ca-ATPase Gene” by Ricardo Escalante and Leando Sastre