Further Findings on BLMash Gene Mutation Identification By Use of

PCR and Gel Electrophoresis in B-Lymphocyte Cells

Lindsey Armstrong (PI), Lynne Davis (LT), Dean Sharrard (PE), Keaton Quinn (DRD)

Abstract:

The BLMash gene mutation codes for Bloom Syndrome (BS) and is highly prevalent amongst Ashkenazi Jews (Ellis et al, 1998). This mutation deletes TAGATTC, and replaces it with ATCTGA at the 2,281^st base pair (bp) on the long arm of chromosome 15 (Ellis et al, 1998). Our experiment was designed to help diagnose individuals with BS and find further possible side effects of affected individuals altered lifestyles. DNA was obtained from homozygous BLMash mutant and wild-type (wt) individuals. We predicted both the mutant and wt primers would bind to wt or mutant DNA at specific temperatures because the primers were designed to complement the appropriate strands (Schochetman et al, 1988). We hypothesized the primers would amplify 371 bp’s of DNA because the primers lengths plus the distance between the primers equal this. PCR and gel electrophoresis were used in attempt to test whether DNA samples were mutant or wt for BLMash which would have been determined by the presence, or absence, of bands at 371 bp’s. To better understand lives of individuals with BS, we ran a sociological experiment. Two members avoided sunlight, as BS patients need to, while two maintained normal lifestyles. To assess our moods we took Seasonal Affective Disorder (SAD) surveys daily. P-values of 0.0053, 0.00000366, and 0.0000000769 showed a significant difference in loss of energy, oversleeping, and carbohydrate cravings between the two lifestyles. Altogether, these findings are key in making advancements in the medical field and positive results in the future could validate this method as a diagnostic tool for identifying the presence of BLMash.

Figure 8: The average recording of seasonal affective disorder (SAD) symptoms intensity that were expressed by both the students living the Bloom Syndrome lifestyle and the students living the normal lifestyle. SAD test surveys containing ten symptoms of SAD were distributed to all group members including 2 that were doing the Bloom Syndrome lifestyle and 2 members that were doing the normal lifestyle. Results for each question were statistically analyzed using Excel. Over a five day period the members of the group were asked to take a survey test once per day and circle the intensity level, scale (0-4), of how these symptoms applied to how they felt throughout that day. T- test was run for every symptom to test if there was a significance between symptoms exhibit by bloom syndrome students and syndromes of the normal students. According to T- test results, there was a significant difference in loss of energy (2), oversleeping (5), and craving for food (7). Anxiety feeling (3), loss of interest in activities (6), gaining unwanted weight (8), and difficulty concentration (9) when t-test was used, did not show a significant difference between bloom students and normal student’s lifestyle. The other symptoms such as feeling depressed (1), social withdrawals (4), and hopeless feeling about anything (10) were not experienced by either bloom or normal students lifestyles.

Discussion

Experiment Summary

Bloom Syndrome is a rare disease that affects 1/50,000 people of Eastern European Jewish ancestry (Liu et al, 2008). Although rare, BS causes many changes within the body. For example, BS causes humans to be born with a narrow face, causing the ears and nose to be very prominent. A short stature, male infertility, and sensitivity to sunlight, causing blisters and reddening of the face, ears, hands, and forearms are also products of the disease. Lastly, and most worrisome, is the early onset of cancer in Bloom Syndrome patients, which is directly associated with the reddening and blistering of the skin that is caused by sun exposure (German et al, 1969). Although sunlight is a major factor in the symptoms of BS, the genetics behind BS stem from a deletion of six base pairs on the long arm of chromosome 15, with an insertion of 7 base pairs in its place (Ellis et al, 1998). Not only does this affect the two amino acids being replaced, but it also causes a frameshift, creating an early stop codon, making the mutant protein abnormally short.

The question we decided to attend to was whether it was possible to create a test using PCR to determine if a patient had the BLMash mutation. To do this we designed three primers. The first was a wild type primer that would attach to the DNA sense strand if the patient had wild-type DNA while another was the mutant primer that would bind if the patient was positive for BLMash. The last primer created was a reverse primer that would attach to the DNA anti-sense strand regardless of whether the patient had the mutation or not so that we could amplify the 371 base pairs of interest (Chen, 2002). When designing these primers we took into account the most favorable PCR conditions such as the temperature and length of each step: denaturing, annealing and elongation, as well as the number of cycles that should be performed (Sambrook et. al., 1989).

A few weeks were spent running PCR and gel electrophoresis strictly on possible positive controls, E. coli and the Lambda virus. These trials were performed not only to achieve optimal conditions for the controls but also to better our understanding and skills of the methods employed in this experiment. After a few trials, results were obtained.

For a negative control wells were run using the usual PRC ingredients but lacking DNA so that no visible bands would form. If bands formed then it would be obvious that there was contamination or some other problem that could be altering results.

While the purification, PCR, and gel electrophoresis portions of the experiment were done in lab, we created a sociological experiment to be done outside of the lab. This entailed half of our group following their normal daily routine while the other half carried out a routine similar to a person with Bloom Syndrome for a week’s time. Those in the group who imitated a Bloom Syndrome routine had to stay away from direct sunlight (German, 1969). Therefore they had to remain indoors as much as possible, wear sunscreen, cover their entire body if going outdoors, and take a route to class that reduced their exposure to the sunlight. Both halves of the group recorded their moods and feelings using questioners that inquired on key aspects of Seasonal Affective Disorder every morning during the week. Seasonal Affective Disorder’s more common symptoms are that of fatigue, depression, and lack of motivation (Eagles, 2003). These symptoms can persist with the lack of light (Rosenthal, 1985). The questions were ranked on a scale from zero to four, zero meaning no signs or feelings associated with a questions and four meaning a question was significantly true. Questions included things such as: how significant were your carbohydrate cravings, your decreased interest in doing work, your lack of energy, your feelings of depression, and your feeling of sleepiness during the daytime hours?

Original Predictions

We hoped to purify 3-8µg of pure DNA from Calu3 and S9 cells using the Generation Capture Column Kit provided by QIAGEN. This expected amount was based off of the vendor handbook that suggests 3-8µg as the average yield.

We expected our results would be similar to those in The Bloom Syndrome Gene Product Is Homologous to RecQ Helicases. These researchers ultimately diagnosed individuals due to the presence or absence of a band on the agarose gel. The presence of bands was associated with having BS but the absence of bands implied cell normality when mutant primers were used. (Ellis et al, 1995)

Our primers were originally designed so that the forward mutant primer would only bind if the mutation was present. The wild type primer is 3’ CGATGTATAGACTGTCCA 5’ and the mutant primer is 3’ CGATGTATAGATTCGTCC 5’. To ensure this, the primers were made to attach at the 3’ end at the site of the mutation. The same general concept was applied to the forward wild-type primer. The reverse primer was designed to bind with both types of DNA 371 base pairs away from the binding site of the forward primers. We predicted this would make the amplified bands 371 base pairs in length.

By amplifying BLMash affected DNA from a Bloom’s Syndrome patient with the mutant primer and running gel electrophoresis we expected to see a band that corresponds with the base pair arrangement of the mutation of 371 base pairs. We thought this would indicate a successful attachment of the mutant primer with the mutated DNA concluding that PCR was successful in determining the possibility that a patient has BS. When running PCR and gel electrophoresis on mutated DNA and wild type primers we predicted no bands would show because the primers would not be capable of binding to the DNA (Ellis et al, 1995). However, we predicted that running PCR on DNA of a normal patient with the wild type primer would result in a band at 371 base pairs.

In regards to our sociological experiment, we predicted that those in our group who followed the routine of a Bloom Syndrome patient would exhibit significant results in each area of the test while the other half of the group would not due to the sun exposure they would still be obtaining (Rosenthal, 1985).

Ultimate Finding and Troubleshooting.

When determining our positive controls we found that running PCR for 35 cycles and with an annealing temperature of 45 degrees Celsius worked best for both the E. coli and the Lambda virus primers. These discoveries allowed us to conclude that our annealing temperature for our positive control, which we decided to be the Lambda virus, would be the previously used temperature of 45 degrees Celsius to assure PCR was working properly and binding was occurring with the primers.

For our experimental assays, no meaningful results were obtained. Several gels contained bands we attribute to primer-dimer (occurring when primers bind to themselves since their sequences are too complementary) due to their appearance below the 100bp mark on the 1Kb ladder (Brownie et. al., 1997). To determine the best experimental annealing temperatures of the wild-type primer, we varied the annealing temperatures, from 42 to 50 degrees Celsius; and, after gaining no results, we began to suspect our initial genomic purification yields were measured incorrectly and the purity values obtained were faulty. Therefore, we decided to re-perform genomic purification on both Calu-3 and S9 cells. EPOCH spectrophotometer reported a 2.4 ng/µL concentration for Calu-3 cells and a negative concentration for the S9 cells. These values were not promising. We suspect the EPOCH spectrophotometer however, we ran another PCR—ran at 44.5,45, and 45.5 degrees Celsius annealing temperatures—with our wild-type and reverse primers to see if we could produce any bands. Again no amplified bands appeared.

At the eleventh hour we received mutant DNA from the Coriell Cell Repository. We had enough time to run one last PCR using our mutant primer at 43 and 44 degrees Celsius (as these were the predicted annealing temperatures we calculated). Unfortunately, once again, gel electrophoresis revealed no amplification.

In all cases, the Lambda positive control band appeared in gel electrophoresis, indicating no issues running gel electrophoresis.

Concerning our sociological experiment, a t-Test was run for every symptom to test if there was a significance difference between the symptoms exhibited by bloom-syndrome-simulating students and normal students. According to T- test results, there was a significant difference in loss of energy, oversleeping, and craving for food. Feeling anxious, loss of interest in activities, gaining unwanted weight, and difficulty concentrating did not show a significant difference between bloom students and normal student’s lifestyle. The other symptoms such as feeling depressed, social withdrawals, and feelings of hopelessness were not experienced by either bloom or normal students lifestyles.

Consequently, we report that there is a correlation between loss of energy, oversleeping, and craving for food and the avoidance of sunlight. These are known symptoms of depression (Kohout et. al., 1993). There is a possibility that these findings coincide with the findings of Rosenthal et. al., who report that light therapy (exposure to sunlight) has an antidepressant affect. Therefore, since patients with Bloom’s Syndrome avoid sunlight to prevent skin irritation, we propose it is possible they are at a greater risk of developing depression.

Future Directions

Since no bands appeared at the 371 base pair mark we suspect two things, either our primers were designed incorrectly or our DNA concentration was too low (Sambrook et. al., 1989). Since no amplification occurred with either our mutant or wild-type primers it is entirely possible they were designed incorrectly. In following experiments we propose re-examining the desired DNA and Primer sequence to see if they match up. If it is found they were not complementary, we suggest making appropriate changes to the primer design.

Conversely, an inadequate amount of DNA may have been used in our PCR cocktails since this is a possible problem that results in no amplification (Roux, 1995). We received 2.4 ng/µL of DNA from genomic purification, meaning we used only 2.4 ng in every PCR cocktail. Etlik et. al. determined in their experiments that adequate results could be obtained from 5 ng of DNA template. Therefore, we suggest—while dealing with the concentrations we obtained—doubling the concentration to 2 µL DNA solution in PCR cocktails.

To confirm the identity of the bands that appear below the 100bp mark of the 1Kb ladder, we recommend running a primer dimer control by running a PCR cocktail containing the usual ingredients but lacking DNA template. The results of this in gel electrophoresis could be a reference of what primer dimer looks like, if primers are indeed annealing to themselves.

To get more accurate and dependable results when dealing with our sociological experiment, we recommend running the trial for a greater amount of time—possible for a full 30 days—and increasing the sample size. According to Dr. Aklilu Zeleke (statistician at Lyman Briggs College of Science) these should eliminate outlying data and verify the correlation between the avoidance of sunlight and symptoms of depression.

If these laboratory techniques were perfected, they could be used as an advanced screen for genetic mutations. This is valuable as genetic sequencing can be expensive whereas PCR is relatively cheap (according to the vendor handbook for the genetic analyzer sold by Applied Biosystems). Given that a hospital or other testing center has access to both a thermocycler and a Genetic Analyzer for genomic sequencing, PCR could be run at a lower cost, easing the monetary strain on patients, hospitals, and the health care system.