Homogeneity of Genome in Homo sapiens and Equus caballus by
Identifying the ATP7B Gene via PCR and Gel Electrophoresis
By: Maddie Barrett, Davin Hami, Maggie McDonald, Allison Vlk, and Hannah Zawisa
LB 145 Cell and Molecular Biology
Monday and Wednesday 12:35 PM
Dr. Douglas Luckie, Anthony Watkins, Alex Strohm, Samantha Thacker
4/21/2017
www.msu.edu/~vlkallis/
Abstract
Written by: Davin Hami
Revised by: Maggie McDonald
Finalized by: Hannah Zawisa
The ATP7B (ATPase copper transporting beta) gene on chromosome 13 is the major contributor to regulating copper throughout the body because it excretes excess copper into the bile and plasma (Van den Berghe et al., 2009). A target segment of ATP7B was amplified and analyzed using allele-specific polymerase chain reaction (PCR) and agarose gel electrophoresis; the experimental purpose was to determine if the gene is present in humans and its horse homolog. We hypothesized that the conserved gene sequences between ATP7B's location in humans and its predicted location in horses will allow for similar primers to be made that will bind to similar locations on the gene, therefore identifying the presence and location of ATP7B in horses (Amvrosiadou et al., 2015). PCR was run on both human and horse DNA, with designed primers for each, and additionally on DNA from the Rz gene of lambda virus as a positive control. To obtain DNA for experimentation, DNA was unsuccessfully purified from cheek cells using several purification methods, and purified horse DNA was received from Dr. Stephanie Valberg, DVM, of Michigan State University. After running the three assays through PCR, they were analyzed using gel electrophoresis. Our results of amplification of the lambda Rz gene was a length of 400-bp similar to the published finding of 394-bp, confirmed by a semi-log plot equation (Kedzierska, 1996). The negative control was a PCR mix without dNTPs. The results of the amplification of the ATP7B in horses produced a 307-bp band, confirmed by a semi-log plot, that was only 1-bp off of its predicted 306-bp band (Wade et al., 2009) Amplification of ATP7B in humans was not successful due to the impurity and low concentration of human DNA and the time deficiency to run further tests. For both human and horse PCR, negative controls were a PCR mix without dNTPs and another without MgCl2, and the positive control was running lambda virus in the gel. ATP7B's presence in both homologs is scientifically relevant because it could prove an evolutionary similarity, which could lead to advances in medical diagnostics and disease testing (Boehm, 1989).
Introduction
Written by: Davin Hami
Revised by: Maggie McDonald
Finalized by: Allison Vlk
The ATPase copper transporting beta gene, or ATP7B, is thought to be present in both humans (Homo sapiens) and in horses (Equus callabus), and both species have symptoms correlated to the gene (Weiss et al., 1999). The ATP7B gene in humans is found on the chromosome locus 13q14.3, which is the long arm of chromosome 13 at position 14.3 (Lee et al., 2011). ATP7B accounts for approximately 80,000 base pairs of the entire human genome, while its protein sequence is 1,411 amino acids long (Tanner, 2008). This gene is susceptible to several mutations, the most prominent being the H1069Q mutation. This mutation is present in 30% to 75% of the Caucasian population (Van den Berghe et al., 2009). The H1069Q missense mutation occurs on exon 14 of human chromosome 13, and it involves a substitution of the histidine protein for a glutamine protein at codon 1069, resulting in a disrupted rate of ATP binding (Kucinskas et al., 2008). In its wild-type form, the ATP7B gene is responsible for removal of excess copper in the body (Gitlin, 2003). Therefore, if there is a mutation in this gene, excess copper builds up which is harmful towards many bodily functions.
The mutations of ATP7B are linked to Wilson's disease, which is a rare autosomal recessive disorder that affects approximately one in every 30,000 people (Pfeiffer, 2007). When a mutation arises to cause Wilson's disease, the results are hepatic and neurological conditions due to an excess amount of copper in the body (Yamaguchi, et al., 1993). Yamaguchi and his colleagues discovered the correlation of mutations to the disease through southern blot analysis, by localizing a complementary DNA region to the region of the ATP7B gene related to Wilson's disease. The disease can cause several symptoms, including: tremor of the hands, rigidity, dystonia (muscle spasms), bradykinesia (slowness of motion), dysarthria (motor speech disorder, difficulty speaking), dysphagia (difficulty swallowing), and difficulties with writing (Huster, 2010). One third of patients suffer from depression, mood swings, and several other psychiatric issues (Huster, 2010). If diagnosed early, a Wilson's disease patient can expect a normal life expectancy, with lifelong treatment of copper chelation (Ferenci, 2003). Chelation is the removal of metals, and Ferenci discovered the necessity of this treatment through clinical neurological examination.
The overall objective of the study was to scientifically examine and determine if both humans and horses share the ATP7B gene, and therefore share the behavioral symptoms correlated to diseases related to ATP7B (such as Wilson's disease). If there are shared symptoms, horses can be used for disease testing and medical diagnostics could be furthered. In order to examine the possible homology between horses and humans, DNA was obtained and purified to be used in PCR, along with designed primers. Hence, another objective was to purify human DNA through cheek cells. To initially understand how PCR works and the importance of each ingredient in the reaction cocktail, the Rz gene of lambda virus was used in experimentation with the objective of reproducing the amplified segment of the Rz gene.
From successful researched experiments, it was originally predicted that we would see a band indicating the presences of the ATP7B gene using wild-type human DNA because the published primers would be able to bind on chromosome 13 between 72,138 to 72,157 base pairs using carefully designed annealing temperatures and methods (Amvrosiadou et al., 2015). We hypothesized that the conserved gene sequences between ATP7B's location in humans and its predicted location in horses will allow for similar primers to be made that will bind to similar locations on the gene, therefore identifying the presence and location of ATP7B in horses (Amvrosiadou et al., 2015). We predicted that designing our own primers using NCBI's primer blast and amplifying the ATP7B gene in horses would yield a 306 base pair band because these primers would bind to the chromosome during PCR and gel electrophoresis, based on testing the human DNA with published primers and temperatures beforehand (Amvrosiadou et al., 2015). We also predicted that our designed primers would bind to base pairs 19,234,860 to 19,234,879 on chromosome 17 of horse DNA because of the similarities to the ATP7B gene in human DNA and subsequent similar methods for each PCR assay (Wade et al., 2009). Furthermore, our predictions and hypotheses were tested and yielded results of homology between horse and humans due to the discovery of the ATP7B gene in horses. However, many more experiments would need to be conducted in order to confirm the presence of the ATP7B gene within the horse genome.
Discussion
Written by: Maggie McDonald
Revised by: Davin Hami
Finalized by: Maddie Barrett
Experiment Summary
Wilson's disease is an autosomal recessive disease caused by several mutations on the ATP7B gene (Kenney et al., 2007). The ATP7B gene is found on the long arm of chromosome 13, at position 14.3 (Paradisi et al., 2014). The mutations are found on numerous exons of the gene, with the most common mutation (H1069Q) being on exon 14 (Van den Berghe et al., 2009). ATP7B is responsible for transportation of copper, a P-type ATPase which maintains copper levels in the body (Katoh et al., 2006). Disruptions in the gene decreases ATP binding, due to a disruption of the WND protein (Hsi et al., 2008). This results in copper toxicity, leading to hepatic and neurological issues. Specifically, the excess copper equates to damage in the liver resulting in cirrhosis as well as brain damage to basal ganglia cells (Desai et al., 2008). The purpose of this experiment is to experimentally prove the presence of the ATP7B gene in Homo sapiens using PCR and gel electrophoresis, as well as finding a similar gene in Equus caballus to determine an evolutionary similarity. We hypothesized that the conserved gene sequences between ATP7B's location in humans and its predicted location in horses will allow for similar primers to be made that will bind to similar locations on the gene, therefore identifying the presence and location of ATP7B in horses (Amvrosiadou et al., 2015). This hypothesis was tested through designing primers for both humans and horses that would bind to purified DNA of both homologs through PCR. After PCR amplification, the assays were run through gel electrophoresis analysis to observe results.
Original Predictions
The ATP7B gene was the target of the research because its mutations can cause Wilson's disease itself, which introduces several behavioral symptoms such as dysarthria (Huster, 2010). For Homo sapiens, the forward wild-type primer used in PCR binds from base pairs 72,138 to 72,157 on exon 14 of chromosome 13 (Weiss et al., 1999). The reverse wild-type primer binds from base pairs 72,432 to 72,453 on the same exon (Weiss et al., 1999). We predicted that performing PCR and gel electrophoresis on wild-type human DNA would amplify the ATP7B gene and produce a 316-bp band because published primers, calculated reactant concentrations, and published annealing temperatures were used that produced successful results (Amvrosiadou et al., 2015).
To determine the presence of the ATP7B gene in an organism that has not been heavily researched, we tested DNA from Equus caballus, or horses. For Equus caballus, the forward wild-type primer used in PCR binds from base pairs 19,234,860 to 19,234,879 on chromosome 17 (Wade et al., 2009). The reverse wild-type primer binds from base pairs 19,235,165 to 19,235,146 on the same chromosome (Wade et al., 2009). We hypothesized that designing our own primers using NCBI's primer blast and amplifying the ATP7B gene in horses would be successful because these primers would bind to the chromosome during PCR and gel electrophoresis and create a 306-bp band, based on results from testing the human DNA with published primers and temperatures beforehand (Amvrosiadou et al., 2015). We predicted that the gene would be present in horses, because of the conserved gene sequences between both homologs, pointing at a potential evolutionary similarity between horses and humans (Wade et al., 2009). We expected the bands produced in gel electrophoresis to be similar in both humans and horses, as the human DNA product is 316 base pairs and the horse DNA product is 306 base pairs.
We predicted the successful amplification of human and horse DNA would result in the presence of the ATP7B gene in both species because of the similar band lengths displayed in gel electrophoresis, due to a similar binding of primers on the respective gene locations (Wade et al., 2009). We predicted that the optimal annealing temperature for gene amplification would be 56°C (Homo sapiens) and 56°C (Equus caballus), because the primers may not have bound to the correct nucleotide sequence if the temperature was too high, so a temperature at the lower end of the typical primer spectrum was expected to be ideal (Green et al., 2015). This is significant because up to 80% of chromosomes found in the gene of Wilson's disease can be identified using this method of analysis (Amvrosiadou et al., 2015). Therefore, if the gene is present in two different homologs, then diagnostic testing on organisms other than humans could be less threatening and has the potential to save human life (Wiegers, 2004).
Results and Ultimate Findings
The lambda Rz gene was amplified using PCR and gel electrophoresis, and a band of 400-bp was discovered on the gel (Figure 1A). PCR and gel electrophoresis were run in order to use a pre-prepared DNA template ladder that has been heavily experimented and published. Running the lambda Rz gene was also to ensure that both tests can be run properly (Kedzierska et al., 1996). Using the full coding sequence of the gene, a 394-bp length was expected, and this was verified to be the correct amplification of the gene by using the semi-log plot. The correct annealing temperature set on the thermocycler for lambda was calculated to be 55°C. When PCR was run at this temperature, a strong band was produced (Figure 1A). The correct length was acquired by a PCR cocktail with an absence of MgCl2.This specific PCR cocktail yielded the most prominent bands when gel electrophoresis was run. This cocktail allowed the forward and reverse primers to correctly amplify the lambda gene (Figure 1A). The cocktail with MgCl2 resulted in no bands present in the gel electrophoresis which was an indication of a failed polymerase chain reaction. Mathematical analysis of the semi-log plot revealed a 392-bp product, which is close to the ideal 394-bp length of lambda, resulting in a practical positive control with a correct amplification of DNA (Figure 1B).
Amplification of the ATP7B gene in humans was unsuccessful due to the failure to purify human DNA through several methods of genome preparation. The first method of genome preparation that resulted in unsuccessful purification of DNA was the Qiagen Genomic-tip 20/G; an analytical gel was run to verify the presence or absence of purified DNA (Figure 2). The second method tested for genome preparation was the Chelex-100 resin beads and another analytical gel was run to check for purified DNA (Figure 3). Finally, the Qiagen Capture Column purification method was used to attempt to purify human DNA and an analytical gel was run to confirm the presence or absence of the properly prepared genome (Figure 4). A band of 316 base pairs in length was expected to be present in the gel. Three PCR cocktails were prepared: one contained a buffer with MgCl2, one contained a buffer without MgCl2, and one cocktail was prepared without dNTPs. The optimal annealing temperature given on the primer vials was 54°C, and this temperature was used during PCR. A band was expected to appear at 316 base pairs in the lane containing the PCR cocktail with MgCl2. However, due to the failure of the DNA purification, there was no purified DNA in any of the PCR cocktails, resulting in no amplification of the ATP7B gene in gel electrophoresis. Although this amplification was unsuccessful, this does not indicate that the ATP7B is not present in humans; further experimentation with PCR amplification using purified human DNA from a research lab is necessary to verify the accurate amplification of this gene and to confirm the homology of the gene amplified in horses.
The ATP7B gene in horses was amplified using PCR and gel electrophoresis. A band of 307 base pairs was observed on the gel (Figure 8A). Purified horse DNA was received from Dr. Stephanie Valberg, DVM, of Michigan State University and was used in the PCR cocktail. Three cocktails were prepared: one contained a PCR buffer with MgCl2, one contained a PCR buffer without MgCl2, and one without dNTPs. The optimal annealing temperature given on the primer vials was 57.8°C, and this temperature was used during PCR. As expected, there was no band found in the well containing the cocktail without dNTPs. The band found in the gel was present in the well containing the cocktail with MgCl2 and was compared next to a 1kb+ molecular weight ladder in order to accurately measure the size of the band in base pairs. Analysis of the migration distance of the band in a semi-log plot verified a 307-bp product which was nearly identical to the expected 306-bp length of the ATP7B gene amplified during PCR (Figure 8B). This suggests that the ATP7B gene exists in horses, although further research and experimentation using PCR and other methods is necessary to confirm the presence of the ATP7B gene in this species.
In the horse PCR and gel electrophoresis experiments, the only band yielded was when the cocktail with MgCl2 was used. However, the positive control that was used only yielded bands when the PCR cocktail didn't contain MgCl2. The inconsistencies within these results point to a problem with the positive control. Therefore, results will need to be validated in further experimentation to ensure that the ATP7B homology, as well as the lambda Rz gene, were amplified properly.
Future Directions
Written by: Hannah Zawisa
One assay that did not produce results was the purification of cheek cell DNA using the Qiagen Blood and Cell Culture Mini Kit. In each attempt, the Epoch spectroscopy showed extremely low purity and concentration of DNA in the sample. The protocol for purifying cheek cells did not account for sample sizes that were too large to fit in the Eppendorf tubes used in the micro-centrifuge. When splitting the samples to account for their size, the concentration of DNA was decreased in each sample, leading to smaller pellets of DNA that were more easily mistaken for supernatant, and removed. In addition, because of the length of the protocol, it took multiple days to finish and required many processes of freezing and thawing. In future attempts, it would be beneficial to run the entire procedure of purification in one period to avoid freezing and to use a larger centrifuge, allowing for full sample sizes to be used instead of splitting the cell solution.
Another assay that was unsuccessful was the purification of cheek cell DNA using Chelex-100 beads. After analyzation through Epoch spectroscopy, the Chelex-100 beads produced very pure DNA, but did not give much yield. This is likely because the protocol required a very small sample of saliva in solution with saline water instead of any stabilizing agent, such as PBS. Because saliva does not contain a multitude of cheek cells to begin with, the small sample size made it even more difficult to obtain a high yield of DNA. If more time was allotted, it may be possible to obtain a greater yield by starting with a larger sample size, that may contain a higher concentration of DNA.
Furthermore, because both of these purification techniques were unsuccessful, the amplification of ATP7B in humans was unsuccessful. A plausible reason for unsuccessful amplification could be that the initial research where the human primers were obtained from used a Multiplex PCR Master Kit that did not have specific contents, so it was impossible to replicate the research completely. In addition, the calculated annealing temperatures were different from those used in the original research, so the primers may have not annealed to the DNA in PCR. In the future, it would be beneficial to research the PCR Master Mix to find exact ingredients and concentrations used and, if more time was allotted, many more cycles of PCR could be ran using both the calculated annealing temperatures, and those used in previous research.
Figures
Written by: Allison Vlk
Revised by: Hannah Zawisa
Finalized by: Davin Hami
A.
B.
Figure 8: Amplification and analysis of the ATP7B gene of Equus caballus by PCR and gel electrophoresis. (A) The 306 base pair target sequence of the ATP7B gene was amplified using the forward primer (Rz1F) and reverse primer (Rz1R). Three PCR cocktails were made. The first contained 7 microliters of 10X PCR buffer without MgCl2, 38 microliters nuclease free water, 1 microliters of both the Rz1F and Rz1R primers, 1 microliters of , 1 microliters of Taq polymerase, and 1 microliters of lambda DNA template. The second cocktail was made with the exact same amounts of each ingredients from the first, except the no dNTPs were added. The third cocktail was made with the exact same amounts of each ingredients from the first, except the 10X PCR buffer contained MgCl2. The three cocktails were placed in the thermocycler and were run at 95°C for an initial 3 minutes. Following this, the samples were cycled 25 times between denaturation at 95°C for 30 seconds, annealing at 57.8°C for 30 seconds, and elongation at 72°C for 1 minute. The 1% agarose gel was made by dissolving 0.4 microliters of agarose powder in 4 ml of 10X TBE and 36 ml of DI water. The solution was heated until completely dissolved and when cooled, 4 microliters of GloGreen dye was added. The final solution was poured into a gel plate in a gel box with dams and a comb in place. When the gel was solidified after twenty minutes, 3 microliters of 6x bromophenol blue loading dye was pipetted into wells 1-4. Then 5 microliters of 1 kb Plus ladder was pipetted into well 1, 7 microliters of horse DNA (without MgClMgCl2 in the PCR buffer) was added to well 2, 7 microliters of horse DNA without dNTPs was added to well 3, 7 microliters of horse DNA (with MgCl2 in the PCR buffer) was pipetted into well 4, and 7 microliters of the lambda DNA was added to well 5 as a positive control. The lambda DNA served as a positive control because it proves that the PCR and gel electrophoresis work properly, due to a past organism's DNA being successfully amplified. The gel was run at 135V for 25 minutes. The gel was placed under a UV light to analyze the presence of the amplified segment of the gene at approximately 400 base pairs. (B) From the gel, a semi-log plot was made by measuring the distance each band of the template ladder traveled from the well. The migration distance (in centimeters) was plotted on the x-axis against the corresponding molecular size (in base pairs) on the y-axis. A trend line was added to obtain an equation that was used to calculate the base pair length of the PCR product of the lambda virus. The equation of the trend line was y = 63054e-1.157x and by plugging in the migration distance of the band, which was 4.6 cm, for x, the base pair length was calculated to be 307 base pairs. When compared to the predicted 306-bp sequence of the ATP7B gene, the semi-log plot equation of the regression line confirmed the presence of the ATP7B gene, because the observed 307-bp length was within error range.