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       The truncation, or removal of a fragment from a gene, of the Disrupted in Schizophrenia 1 (DISC1) gene accounts for 1.8% of people affected by bipolar disorder happening at the C-terminal domain on chromosome 1q42 (Hodgkinson et al., 2004). This gene is present in humans and other animals. We hypothesized that the DNA extracted and purified from Homo sapien (human) saliva using QIAGEN Generation Capture Column Kit will code for the DISC1 gene when performing polymerase chain reaction (PCR) methods with primers generated by researcher Kazuhisa Maeda and her colleagues (2006). The products were determined with agarose gel electrophoresis and a band 287 base pairs was amplified and detectable using UV light, although the base pair lengths were predicted to be 331 (Maeda, et al., 2006). Since there is genetic evidence that some avians have DISC1, we also hypothesized that Aptenodytes patagonicus (King penguin) could code for the DISC1 gene given their genetic similarities (Sanchez and Ponting, 2011). A PCR cocktail was prepared with designed primers for the King penguin from Life Technologies Corp. and Thermofisher; a band of 245 base pairs were amplified when using gel electrophoresis and UV light when a base pair length of 252 was predicted. Both agarose gel experiments were compared to a DNA ladder with known base pair lengths. To insure all materials were functional, we used the 16S Escherichia coli (E. coli) as a positive control. Connecting one gene between human and bird, more evolutionary links could develop in the world of ecology. The designed primers were also run with the human DNA to further confirm possible genetic linkages between human and King penguin. Macaca mulatta, (rhesus monkey) and Mus (mouse) homologous DISC1, are an example of two distinct organisms that share this gene (Bord, et al. 2006). By cross species analysis, we could expand on an evolutionary scale disease-associated genetic variation within the King penguin and interpret data for advancements in human treatment and medicine for bipolar disorder.

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Summary of Experiment

       Bipolar disorder is a disease that is characterized by a truncation, or shortening, of the C-terminal of the DISC 1 gene that is found in the hippocampus of the brain (Hodgkinson et al., 2004). The DISC1 wild type gene functions as a molecular scaffold that interacts with proteins that are used for neurite growth, signal transduction, and multiple other signaling behaviors that are important for cellular function (Clapcote et al., 2007). Signal transduction is the process of translating information that is attached to the receptors on the outside of the cell (Clapcote et al., 2007). Once the gene is truncated, signal transduction is altered and the cells are unable to grow and communicate (Clapcote et al., 2007). Researchers often look to DISC1 to determine possible neurological causes of diseases like anxiety, schizophrenia, and bipolar disorder (Ellis and Soanes, 2012). We used PCR to research the existence of the DISC1 gene that may cause bipolar disorder in King Penguins in order to explore similarities in genome makeup between penguins and various organisms. We hypothesized that if published DISC1 human primers were mixed with human DNA in a PCR cocktail, then an amplified product of 331 base pairs would result (Maeda et al., 2006). It was also hypothesized that if King Penguin DNA was mixed in a PCR cocktail with primers we designed based on the DISC1 sequences of various organisms such as homo sapiens (humans), Macaca mulatta (Rhesus monkey), Canis lupis (gray wolf), Capra hircus (goat), and Bos taurus indicus (zebu cattle) then we would expect DNA amplification to be successful and the base pair product to be similar (252 bases) to the base pair product length of the human PCR experiment due to the strong phylogenetic relationships that exist between penguins and the organisms used to design the primers.

Original Predictions

       We predicted that the published forward primer 5’-(GAC TCG CTG AGG AGA AGA AAG)-3’ and reverse primer 5’-(GGT TGT TAA CAG AGG CAC GC)-3’ designed for the Disrupted in Schizophrenia 1 wild type gene sequence at exon 1 would anneal to human DNA due to correct temperature selection and accurate PCR thermocycler temperatures (Maeda et al., 2006). It was also predicted that the primers we designed (forward primer 5’-CAC TGC TCA GGA CAG CTT GC-3’ and reverse primer 3’-GCA GCT ACA GAA AGA AAT CG-5’) based on the DISC1 homolog sequences found in Macaca mulatta (rhesus macaque), Canis lupis (gray wolf), Capra hircus (goat), Bos taurus indicus (zebu cattle) and Homo sapiens (human) would anneal to Penguin DNA when mixed in a cocktail due to the correct calculations of annealing temperatures based on the nucleotide makeup of the primer (Rychlik et al., 1990).

Summary of Results and Ultimate Findings

Our control experiment in which 16s rDNA of Escherichia coli was amplified using Polymerase Chain Reaction resulted in a product of 1556 bases when it was run through a gel. This success allowed us to confirm that the ingredients used in the cocktail were functioning properly, and that if our experiment failed it would be due to faulty primers or incorrect annealing temperatures. These ingredients include the dNTPs (free nucleotide bases), double distilled water, taq polymerase, MgCl, and the PCR buffer. For the primary experiment, numerous cycles were run with three different combinations of primers and DNA. These combinations were the published primers (Maeda et al., 2006) being run in a PCR cocktail with human DNA, our designed primers being run with human DNA, and our designed primers being run with King Penguin DNA. When run through a gel, the Maeda published primers and the human DNA PCR experiment yielded only one band too large to move out of the well (Figure 5). This contradicts our original hypothesis, as we stated that the amplified product should run through the gel and be around 331 nucleotide bases long (Maeda et al., 2006). As published primers and temperatures were used for this experiment, there are a few explanations as to what may have occurred. Since human spit was purified to extract the DNA used for PCR, it is possible that the supernatant wasn’t completely drained and discarded during the purification process, which is an absolutely essential step (Liu et al, 2000). This would have left other molecules/impurities attached to the DNA, and as a result the PCR product would’ve been too big to run through the gel. Furthermore, the primers we designed based on a published DISC1 PCR research experiment could have been translated incorrectly during the ordering process, causing the primers to not anneal to the DISC1 sequence and amplification being unsuccessful as a result. This would result in a band too large to run through the gel as well. When the purified DNA was originally run through the EPOCH machine it detected .62 nanograms of DNA, confirming that we had the DNA necessary for the PCR reaction. For the King Penguin and designed primer PCR experiment, a variety of trials involving different temperatures and various dye to DNA ratios were executed. After a number of adjustments, a temperature gradient of 49 degrees celsius to 54 degrees celsius was settled on by using the equation (Tm =81.5 + 16.6 log [K+] + 0.41 (%GC) - 675/N (Heckler and Roux, 1996). When the amplified penguin DNA product of the described experiment was run through a gel and visualized under ultraviolet light, the bands were extremely light in comparison to the 1-KB Plus DNA Ladder that it was run next to (Figures 4). To confirm that the bands were made up of the expected amplified product and not extra ingredients such as dye, a semi-log plot was created that included the molecular weight for fragments of the DNA Ladder (y axis) vs. the migration distance of the fragments from the entrance of the well (x axis) (Figure 4). Using the resulting exponential equation, the migration distances for the light bands of amplified DNA were plugged into the equation and outputted the band lengths of the amplified product. The calculated band length was 245 base pairs for the successful band. This result supported our original hypothesis, as this length was around the predicted band length of 252 base pairs for this PCR experiment. One possible reason that the penguin DNA amplified product length wasn’t closer to that of the human amplified product length (331) is because the primers we designed to anneal to penguin DNA were made from DISC1 homologs in various organisms and not the DISC1 gene located in penguins. This means that there are likely discrepancies between the DISC1 sequence in humans and the DISC1 sequence in penguins. It is also entirely possible that the designed primers don’t completely compliment any part of the DISC1 gene, and so complete annealing isn’t possible. When primers don’t anneal completely then weak bands may result; this can be seen in figure 4. Weak bands may also indicate a primer dimer, in which the forward and reverse primer anneal to each other instead of the target strand. For the third combination, our designed primers were mixed in a PCR cocktail with human DNA purified from spit. After the cocktail had gone through normal PCR procedure and had its amplified products separated through gel electrophoresis, an extremely light band was observed (Figure 6) just as it did with the amplified penguin DISC1 gene. A similar strategy was used, with the semi-log plot and equation being created. A base pair length of 287 was calculated, compared to the predicted human DISC1 amplified product of 331. This relatively successful amplified product would suggest that the designed primers were quite functional while the published primers we used to amplify DNA were faulty (due to the inability of the amplified human DNA product to run through the gel). Our research implicates that the DISC1 gene can potentially be found in a various number of organisms, and the presence of genetic diseases such as bipolar disorder can be explored within the genome of these organisms. Many scientists have already amplified the DISC1 gene in other animals in order to find the effects a mutated DISC1 gene sequence can have. One study done by researchers at Columbia University Medical Center in New York tested the mutated DISC1 gene and the effect it can have on memory loss in mice. They accomplished this by amplifying exon 6 of a DISC1 allele, and in doing so were able to detect a 25 base pair deletion in the DISC1 sequence (Koike et al, 2005).

       One possible weakness in our experimental design could be the fact that we didn’t actually have the mutated form of the DISC1 gene. If we had been able to acquire DNA of a human who had bipolar disorder, then we could have further strengthened the link between bipolar disorder and the DISC1 gene. Additionally, if we successfully designed primers based on mutated DISC1 sequences in various organisms (humans in particular) and successfully amplified penguin DNA based on these primers, then that could have serious implications about penguins and bipolar disorder. Of course, the DISC1 gene mutation is far from the only reason bipolar disorder exists, as it can be caused by multiple different variables including environmental effects and other genetic discrepancies (Leahy, 2007). Another possible experimental weakness is that when designing primers, we only compared the DISC1 wild type sequences of 5 organisms. If we had used a larger number of organisms, then we may have been able to design primers that had a higher chance of amplifying the penguin DNA completely, or to a similar base pair length that should have resulted in the human PCR experiment. Near the end of our research, one serious technical difficulty that we had pertained to the running of the bands through a gel. The ladder was not showing up as dark as it used to be, and neither was the amplified DNA. This could be due to the reasons stated in the Summary of Results section, that the designed primers didn’t completely compliment with the penguin DNA. But, it could also be due to the fact that we changed our gel staining solution from GloGreen to SybrSafe. Unfortunately, this was due to shortages in GloGreen, so there was no way to test whether or not this is a likely possibility.

Future Directions

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       If there were time allotted for more experimentation, failed assays would be analyzed and troubleshooted, even though the amplification of the DISC1 gene was a success overall. The main salient issues were the weak bands in both PCR experiments involving designed primers, slightly incorrect predicted band lengths for the human and King penguin genome, and failed priming for our positive control. Weak bands mean the primers partially annealed during the PCR process. Some primer dimer attachment occurred when the primers annealed to themselves and not the desired DNA template. To address this problem, the designed primers would need to be redesigned with a greater number of guanine and cytosine bases at the 3’ end to enhance binding specificity rather than primers that end with adenine or thymine. Avoiding long runs or strings of a single base can also produce an optimal product (Apte and Daniel, 2009). Since the predicted base pair lengths were not formed, the sequences we created in BLAST gave unintended homologies which resulted in false priming (Apte and Daniel, 2009). Comparing DISC1 sequences of multiple avians and organisms from the arctic to design primers for penguin DNA would increase the chances for a distinct band size in the gel. The positive control experiment worked only with the E. coli bacteria broth. The concentration of the cultured bacteria cells were not concentrated enough in order for the primers to anneal, and as a result they created a faint primer dimer band. Increasing the concentration of cell cultured bacteria would most likely create a more distinct band. Adjusting temperatures could produce a better band as well, however, high annealing temperatures could cause the primers to bind inefficiently (Yuryev, 2007). Once this experiment is completed, further experimentation could include the yeast 2-hybrid test. The 2-hybrid test calculates the number of proteins that interact with the genes receptors as well as how those proteins interact with the receptors, which would help in understanding receptors and proteins actually involved in the mutated genomic sequence (Millar et al., 2003). Each protein is important when it interacts with a gene, and could possibly be linked to a behavior that is associated with that gene. This test could eliminate the wild-type gene proteins interact with, and conclude that proteins that interact with the mutant type gene are responsible for a behavior that is studied (Millar et al, 2003).

Figure

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Figure 1: PCR Bands for amplified Designed Primers analysis using gel electrophoresis. A. Lane MW contained 7 ul of the 250ug 1-KB Plus DNA Ladder, and 3 ul of the blue loading dye which is ~12,000 base pairs long. Bands are amplified throughout the gel. Lane 2 through 7 contained 7uL of purified human DNA cocktail and 3ul of the blue loading dye, and showed bands at 287 bps. Lanes 2 through 7 contained 2ul of the designed forward primer 5’-CAC TGC TCA GGA CAG CTT GC-3’and 2ul of the designed reverse primer 3’-GCA GCT ACA GAA AGA AAT CG-5’. The cocktails for lanes 2 through 7 were run through at a gradient of 49 degrees C to 55 degrees C.. Each cocktail was run in a Bio-Rad T100 Thermal Cycler programmed for 35 cycles. A gel was made with 5g of .8% agarose powder which was mixed with 100 ml 1x TBE buffer solution (Tris, Borate, EDTA) and Green Glo loading dye into each well along with the pipetted cocktail in all 5 lanes and run at 150V to show the bands. B. A semi-log plot for the 250ug/ul 1-Kb Plus DNA ladder. The migration distance (cm) from well to band of was portrayed by the x-axis and x-value, in respect to the molecular size was portrayed by the y-axis. The equation was obtained for analyzing the base pair length of the HUman DNA R2 value .9952 represented the best fit for the trend line for the distance each band travelled. Lanes 2-4 predicted base pair length was 331 bps.