Our Research
Detection of 1510bp
Band Using PCR, Gel Electrophoresis to Diagnose E6V Sickle Cell Anemia in Human
CALU-3, BM Cell DNA
Tyler Ash, Kristina Knirk, Nicole Arcy, Jonathon Walters
Abstract
We hypothesized that two primers, complimentary to wild-type and mutant human genomic DNA for the E6V mutation, would successfully discern between these genotypes under optimal primer design and annealing temperatures.Primer specificity will permit amplification via PCR only if proper binding to complimentary DNA occurs (Wu et al, 1988). Gel electrophoresis was conducted on PCR products to reveal the presence/absence of bands 1510bp in length, indicating the presence/absence of the E6V mutation. With an increase of DMSO and MgCl2, bands at 1510bp were observed for mutant and wild-type DNA. The purpose was to create an assay that can quickly and accurately reveal the presence/absence of sickle cell in individuals (Saiki et al, 1985). A successful assay can be implemented as a genetic diagnostic for prenatal testing, facilitating improved quality of life and decreased mortality rate by early detection benefits and programs (Vichinsky et al, 1988). Sociological surveying of MSU Lyman Briggs students investigated if negativity associated with genetic diseases causes students to be more pessimistic regardless of knowledge of sickle cell anemia (Marteau et al, 1997). This hypothesis was supported, but no correlation was found. Additionally, four subjects consumed sickle cell diets for two weeks, tabulated costs, and compared them to standard diet costs, predicting that sickle cell diets cost more because they require more protein, vegetables, and fruits priced higher than cheaper and unhealthier food eaten in a typical diet (Jetter et al, 2006). This prediction was also supported by the data.
Introduction
Sickle cell anemia is an autosomal recessive genetic disease affecting approximately 5% of the current human population. It is characterized by the sickling of red blood cells under oxygen stress (Bender et al, 2003). Red blood cells contain approximately 250,000,000 hemoglobin proteins (Campbell and Reese, 2008). Two of the four globular protein subunits that make up a hemoglobin protein are manufactured from the β-globin (HBB) gene located on the short arm of chromosome eleven. This gene sequence is 1606 base pairs in length and the resulting protein is 146 amino acids in length (Ashley-Koch et al, 2000). In the United States, 60-70% of sickle cell anemia cases are caused by a single nucleotide polymorphism on codon six of the β-globin gene (Bender et al, 2003). The mutated sequence consists of a thymine replacing an adenine (GAG to GTG), resulting in the amino acid production of valine instead of glutamic acid (Wu et al, 1988; Saiki et al, 1985). Subsequently, the synthesized β-globin protein's structure, in deoxygenated form and on opposite sides of the hemoglobin molecule, assumes a shape with a partially hydrophobic zone that has a propensity to polymerize with the hydrophobic zones of other β-globin subunits. Consequently, hemoglobin molecules can crystallize into fibers, greatly reducing oxygen carrying capacity and resulting in damaged membranes of red blood cells whose structures have distorted into rigid, crescent shapes (Pauling et al, 1949).
A variety of health complications such as anemia, stroke, vaso-occlusive crisis, splenic sequestration crisis, aplastic crisis, hyper haemolytic crisis, and damage to multiple organs can ensue when red blood cells sickle (Adams et al, 2001; Bender et al, 2003; Mills, 1985; Powell et al, 1992). Sickled cells can accumulate and block blood vessels, resulting in acute and chronic pain (known as sickle crises) as well as possible damage to any organ of the body. Vision problems, for example, can occur from this vaso-occlusive crisis in the small blood vessels in the eyes. Damage to the spleen caused by the sickle cells often leads to increased infection rates. Some treatments for this disease include painkillers for sickle crises, antibiotics for infections, and hydroxyurea to lower the severity and the frequency of sickle crises (Bender et al, 2003).
Improved quality of life and decreased mortality rate can be promoted from early detection of the disease (Vichinsky et al, 1988). This study aims to develop an assay that can accurately, cheaply, and quickly reveal the presence/absence of the E6V mutation in individuals (Wu et al, 1988; Saiki et al, 1985; Waterfall et al, 2001). Through the use of polymerase chain reaction (PCR), the amplification of a targeted region of the HBB gene will indicate the presence/absence of the E6V mutation in wild-type and mutant type individuals (Wu et al, 1988).
PCR is an amplification technique in which, initially, the hydrogen bonds of a DNA sample are denatured by exposure to ~95° C. The temperature is then lowered to ~50-65° C to allow oligonucleotides to anneal to their complimentary portions of DNA at temperatures dependent upon the primers' contents. Next, the temperature is raised to ~68-72° C so that Taq polymerase can attach and extend in the 3' directions, effectively replicating the remaining DNA strands. These three steps are repeated numerous times and, if the primers keep successfully annealing to their respective sites, will yield carbon copies of the nucleotide sequence between the two primers at an exponential rate (Saiki, et al 1988).Primers can therefore be designed to selectively anneal to DNA with or without certain characteristics. The assay developed here targeted the E6V mutation (a single nucleotide polymorphism) and the two primer sets were designed to be complimentary to wild-type and mutant type DNA. The Yaku primer design technique was employed in order to increase efficiency in annealing and therefore genotype discrimination (Yaku et.al, 2008).
The products of PCR were inserted into agarose gels to undergo electrophoresis in which DNA, due to its net negative charge from its spinal phosphate group, migrate from the negative end of the gel to the positive end. Shorter DNA fragments migrated quicker and farther than longer ones; the fragment lengths contained in PCR products were determined by comparison to an included DNA ladder of predetermined band lengths. The two forward primers, complimentary to wild-type and mutant DNA for the E6V mutation, will be able to successfully discern between these genotypes due to specificity permitting amplification during PCR if proper binding to complimentary DNA takes place (Wu et al, 1988).After analysis via agarose gel electrophoresis and UV transillumination, a band 1510 base pairs in length will be detected only if mutated DNA undergoes PCR with the complimentary mutant primer or wild-type DNA undergoes PCR with the complimentary wild-type primer.
The idea of genetic disease can carry with it connotations of determinism, negativity, and little or no availability of preventative or combative action plan.It has been suggested that the term "genetic", or being told one possesses a certain gene, bears connotations of fate or certainty (Marteau, 1997).The current study augments that idea by observing how knowledge of sickle cell anemia affects emotional response to a hypothetical diagnosis of it. Individuals with sickle cell anemia must follow a diet including high levels of fluid intake, protein or omega-three fatty acids, and five to nine servings of green, red, and yellow vegetables, fruits, or juices that are rich in antioxidants (Ohnishi et al, 2000).An additional study is being employed in which four participants record costs of eating a sickle cell anemia diet for one week as well as one week with their typical eating habits to see if the sickle cell diet will cost more than a typical diet because the sickle cell diet requires more protein, vegetables, and fruits priced higher in comparison to cheaper and unhealthier food eaten on a typical diet (Jetter et al, 2006).
Methods
Primer Design and DNA Acquisition
Two forward primers and one reverse primer were designed. The two forward primers were complimentary to either MT or WT DNA and the reverse primer complimentary to downstream MT and WT DNA. The forward primers were created with intentional base pair mismatches at the 3' end (Yaku et al, 2008). The forward MT primer was designed to bind to the mutation at the 19th base pair and the reverse primer was designed to bind 1510 base pairs away from the forward primers. For the assay, Wprimer (forward WT primer):5'-ACCACGTAGACT GAGGTCT-3', Mprimer (forward MT primer): 5'-ACCACGTAGACTGAGGTCA-3', and Rprimer (reverse primer): 3'-TGCCAAAGTGATGGGCC-5'. Using the Oligonucleotide Properties Calculator, Wprimer was calculated to have an annealing temperature of 55.7oC, Mprimer 56.0oC, and Rprimer 56.0oC (Kibbe, 2007). Based on these theoretical temperatures, an annealing temperature of 53.0oC was deemed theoretically favorable and experimental temperatures were based on this.
Following the manufacturer's protocol, DNA purification was performed on cultured wild-type CALU-3 cells provided by Lyman Briggs research facilities at Michigan State University. The mutant DNA (200ng genomic DNA) was obtained from the Kohn Lab in the Department of Microbiology, Immunology, and Molecular Genetics at UCLA in California.
PCR and Gel Electrophoresis
PCR was conducted on mutant and wild-type DNA and its products run through agarose gel electrophoresis. All cocktails consisted of 40µLnuclease-free H2O, 5µL 10X Thermopol buffer, 1µL 10mM dNTPs, 1µL 100uM MT or WT forward primer, 1µL 100uM reverse primer, 1µL MT or WT DNA, and 1µL Taq polymerase. Later experimental cocktails contained one of three additions: 3µL MgCl2, 3µL DMSO, or both.
In the Labnet thermocycler, all cocktails underwent initial denaturation at 95oC for 5 minutes and final extension at 72oC for 5 minutes. Cocktails without MgCl2 and DMSO alterations experienced 30 cycles of denaturation at 95oC for 45 seconds, annealing at 45/50/55oC for 30 seconds, and extension at 72oC for 45 seconds. Cocktails with added MgCl2 and DMSO underwent 25 cycles with annealing temperatures at 44.5/52/56oC.
A 2% agarose gel was created by mixing 0.8g of agarose powder, 40mL of 1X LB buffer, and 1µL of ethidium bromide. For each gel, PCR products were mixed with 1µL of bromophenol blue loading dye. A 1X Kb molecular ladder was used. Approximately 217 volts of electric current ran through the gel for 35 to 40 minutes. The UV transilluminator revealed migrated products. Bands were compared to the molecular ladder to determine length.
"Living Knowledge of Sickle Cell Anemia" Experiment
The sociological experiment was two-fold: 1) subjects followed a sickle cell anemia friendly diet for two weeks and then, within subjects, compared costs to a normal diet, and 2) surveys probed student knowledge of the disease and emotional reaction to a hypothetical diagnosis of it through genetic testing. The diet included high levels of fluid intake, protein or omega-three fatty acids, and five to nine servings of green, red, and yellow vegetables, fruits, or juices that are rich in antioxidants (Ohnishi et al, 2000). The four present researchers participated in this diet for two weeks, followed by two weeks of regular eating. The costs of eating regular food versus the diet food for each individual were calculated using an online food price list, totaled, and compared using a t-test.
The survey consisted of ten multiple choice/true-false questions and one opinion question pertaining to sickle cell anemia. It was administered to students in the Lyman Briggs residential science college at Michigan State University. The opinion question appeared equally at either the beginning or end of the survey, controlling for the possibility of opinion being influenced by exposure to knowledge contained in the other ten questions. The opinion question asked, "How would you feel if you were diagnosed with sickle cell anemia?" Subjects answered using a five-point scale: 0 = very pessimistic, 1 = slightly pessimistic, 2 = neutral, 3 = slightly optimistic, 4 = very optimistic. One point was awarded for each correct MC/T-F answer. The correlation between these results was analyzed with a line of regression in order to compare knowledge of the disease with degree of pessimism/optimism following a hypothetical diagnosis of sickle cell anemia.
Results
PCR
Running PCR on the acquired DNA using Wprimer and Mprimer successfully amplified products of the intended base pair length (1510bp) from the HBB gene located on chromosome eleven (Figure 6). For these results to be obtained, concentrations of magnesium (in the form of MgCl2) and dimethyl sulfoxide (DMSO) were added to the cocktails. Amplification was present with non-specific binding at 45⁰C and 50⁰C for Wprimer with wild-type DNA (Figure 1). Bands of base pair length 1510 were prominent when the magnesium concentration was increased to 5% for mutant DNA with Mprimer at 44.5⁰C (Figure 2). An increase in DMSO resulted in faint non-specific binding at 44.5⁰C for wild-type DNA with Wprimer (Figure 2). This did not, however, show a prominent amplification of the desired base pair length.
For both wild-type and mutant DNA (with respective primers), primer-dimer was present at all temperatures (44.5⁰C, 52⁰C, 56⁰C) with increased concentrations of magnesium, and also for separate replications at the same temperatures with increased concentrations of DMSO (Figure 2). In trials with increased concentrations of both DMSO and magnesium, primer-dimer was present for mutant DNA with its respective primer at 44.5⁰C (Figure 3). A product with base pair length of approximately 250 was present for mutant type trials with increased magnesium concentration at 44.5⁰C and 52⁰C (Figure 2), and with increased DMSO and magnesium concentrations at 44.5⁰C and 56⁰C (Figure 3). The DNA purification was conducted in order to extract and purify DNA from human CALU-3 cells, resulting in approximately 80% DNA.
"Living Knowledge of Sickle Cell Anemia" Experiment
On average, students were more pessimistic than optimistic when faced with the hypothetical situation of being diagnosed with sickle cell anemia. No correlation was found between degree of pessimism and knowledge of the disease with an R2 value of 0.026 (Figure 4). Students demonstrated an average knowledge score of 7/10 (~72%) on the quiz portion. The findings of the diet showed that more money (~$29) was spent on a sickle cell diet compared to that of a regular diet. α = 0.05 was used as the accepted value of significance. For participants one and four of the diet there was no significance found between the cost of the two diets (p = 0.064, p = 0.061, respectively). For participants two and three a significant difference was observed between the costs of the two diets (p = 0.039, p = 0.021, respectively) (Figure 5).
Discussion
Original Predictions
We predicted that by using PCR to amplify DNA from the HBB gene for both wild-type and mutant alleles and then using agarose gel electrophoresis on the products, we would detect the presence/absence of the respective alleles. We predicted that our primers would be able to discern between wild-type and mutant type DNA for the E6V mutation, producing bands 1510bp in length based on techniques used in previous research (Wu et al, 1988; Saiki et al, 1985; Waterfall et al, 2001).Predetermined PCR and electrophoresis results for lambda DNA obtained in lab were to be included in the assay as a control.
We predicted that the sickle cell anemia friendly diets requiring increased quantities of healthy food (fruits, vegetables, antioxidants, etc.) would cost more when compared with a regular diet based on previous research; for a two-week period the healthy food cost $34 more than the standard market basket (Jetter et al, 2006). We also predicted that students will tend to have more pessimistic outlooks given a hypothetical diagnosis of sickle cell disease regardless of knowledge of the disease because genetic diagnostics for diseases tend to have deterministic and negative connotations (Marteau, 1997).
Results and Ultimate Findings
Assuming the designed assay works, the obtained value of this research lies in the test's swiftness and reliability in determination of an individual's HBB genotype. The crux of a comprehensive newborn hemoglobinopathy screening program that is effective in decreasing the mortality rate in children with sickle cell disease lies in early detection via quick and reliable testing. At first, the PCR process was unable to amplify the target DNA regions with the designed primers and annealing temperatures used. However, after adding 5% DMSO and MgCl2 to the cocktails and slightly changing the PCR temperatures, bands 1510bp long were detected for both mutant and wild-type DNA.
The notion of genetic illness conveys for many people negative feelings of being unable to prevent, control, or treat a disease with a genetic cause. Previous studies suggest that the term "genetic", or being told one possesses a certain gene, bears connotations of fate or certainty (Marteau, 1997). Our experiment extended from this research by observing how knowledge of a disease correlates to emotional response when hypothetically genetically diagnosed with that disease. According to the data collected, there were higher numbers of more pessimistic outlooks regardless of knowledge; however, no statistical correlation was found (Figure 4). The results of the diet correspond with previous research in that the costs for the sickle cell diet were an average $29 higher than the regular diet (Figure 5A), though only two of the four subjects had a statistically significant difference in cost (Figure 5B).
Future Directions
The designed PCR assay and primers were successfully able to amplify mutant and wild-type DNA. However, the bands observed were often very faint, and primer-dimer/non-specific binding was also present. In order to produce more distinct gel results, the concentrations used in the PCR cocktails and the annealing temperatures used in the PCR amplification can be varied. Also, more cocktails can be made using DMSO and MgCl2 until the optimal PCR conditions can be established. Better primers can also be designed in such a way that will eliminate primer-dimer/non-specific binding and only allow for the correct amplification of target DNA. One extension of this research can be to apply the developed assay to larger sample sizes of known genotypes in order to further establish its accuracy.If the assay proves acceptably reliable from this, then it can be distributed world-wide for mass testing of all ages, especially prenatally, as well as for any other investigations, like to spatially and temporally correlate E6V frequency with incidences of malaria.
The sociological aspects could also be extended. The average score for the knowledge portion of the survey was higher than anticipated. This could be amended by adding more difficult questions, not only changing the average but also giving a broader range of scores, increasing the validity of the experiment. In addition, the survey distribution could be extended to other residential colleges within Michigan State University, or even further by including other colleges.Also, the diets could be extended to one month each. By extending the time frame a more accurate representation of cost differences could be generated.
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