Genotypic Identification of SCA due to the HbS Mutation in

Human Epithelial Cells via AS-PCR

By: Jasmine Costello, Bobby Dipietro, Kyle Hutchinson, and Sarah Newman

LB 145: Cell and Molecular Biology

Tuesday and Thursday 4:10 PM

Dr. Doug Luckie, Allison Chan, and Emily Schmitt-Matzen

Week 15, April 23, 2013

Abstract

The HbS substitution mutation of the HBB gene causes sickle cell anemia, a disease that causes red blood cells to become sickle-cell shaped which prevents oxygen from being properly carried throughout the body, as well as, creating blood clots. Allele-specific polymerase chain reaction (PCR) was used to determine if the mutant-type DNA or the wild-type DNA contained the HbS mutation (Ye et al, 2001). It was hypothesized that by creating two different forward primers, one with and one without the mutation, the mutant primer would bind to mutated DNA, causing a band to form on the electrophoresis gel at 229 base pairs (bp). This would diagnose sickle cell anemia (Waterfall et al, 2001). Through many trials of PCR, it was found that controls, cited from The US National Library of Medicine National Institutes of Health, that control primers, with the sequence  MTFDP 5’-CAGTAACGGCAGACTTCTCCA-3 and MTRP 5’-GGGTTTGAAGTCCAACTCCTA-3’ would not anneal to the IB3-1 sequence because of a point mutation located on the primer and not on the DNA template (Waterfall et al, 2001). Also, when designed primers, formatted to anneal to both sickle cell DNA template, as well as, IB3-1 DNA template, were ran through PCR and gel electrophoresis, the results were indeterminate due to time constraint.  To understand the social implication of sickle cell, where sickle cell patients are often seen as “drug abusers” and not as actual pain patients, subjects wore a shirt labeling them as a sickle cell patient. The reactions of the public from four locations: Brody cafeteria, a Biology lecture, an Organic Chemistry lecture, and Grand River Avenue were recorded and ran through a t-test statistical analysis. A t-test value of 2.53642716 and a p-value of .0113 were obtained. As hypothesized, based on the significant p-value, it was found that patients living with sickle cell are judged upon first impressions by the general public. By creating PCR assays to diagnose sickle cell anemia, scientists and doctors can work together to help expand the lifespan of someone living with sickle cell anemia, as well as, reduce symptoms over a patient’s lifetime.  (Oringanje et al, 2009).

Discussion

Experiment Summary

Sickle cell anemia is an autosomal recessive disease that is caused by the HbS mutation, in the β-hemoglobin DNA sequence. The mutation is a substitution of a thymine for an adenine at the 30th nucleotide of the normal β-hemoglobin DNA sequence. In turn, the mutation causes the irregular “sickle” shape of red blood cells in humans. This “sickle” shape of the red blood cells decreases the amount of oxygen that each cell can carry and transport to the rest of the body, as well as increases the risk of blood clots. Symptoms include, but are not limited to: hypoxia, jaundice, pain crises, swelling of the hands and feet, chronic fatigue, migraines, deterioration of the retina, infections, stroke, and death (Driscoll, 2007). There is currently no cure, but treatments to manage symptoms are available.

A polymerase chain reaction (PCR) test can amplify a mutated HBB gene, easily diagnosing sickle cell anemia. By pipetting a mixture containing amplified DNA onto a gel electrophoresis test, it can be  determined whether the DNA is positive or negative for sickle cell anemia, based on the locations of the DNA in the gel. Overall, based on the need for genotypic identification of sickle cell patients, it was hypothesized that a forward mutant-type primer, put through PCR and ran against both wild-type and mutant-type DNA templates in a gel electrophoresis test, would anneal to only to the mutant-type DNA template, creating a band on the gel at 267 base pairs (Waterfall et al, 2001) (Figure 2).

Another portion of the experiment was to live in the life of someone with sickle cell anemia, as well as, address the social stereotype found in medicine in which sickle cell patients in a pain crises are compared to “drug seekers”, a term used to denote people who visit emergency rooms only to obtain pain medicine to get high (Adamson, 1995). In the experiment, designed “sickle cell T-shirts” were created and tested around four different locations at Michigan State University of East Lansing: Brody Cafeteria, Grand River Street, Biology Lecture and Organic Chemistry Lecture. The T-shirts were labeled “I have sickle cell anemia” on the front and “I am not a drug abuser” on the back. Two subjects wore bright red sickle cell shirts with blue labeling, while two other subjects wore black shirts along with bright pink lettering. In each location, one subject, with a sickle cell shirt on, would be recorded by another subject, without a sickle cell shirt, trailing far behind to not attract attention to the test subjects. Control groups, subjects without the sickle cell shirt on, were also recorded in this manner. It was predicted that 55% of the population would shows signs of negative reaction, such as:  acts of verbal or visual disgust, physical gestures, such as pointing or laughing (CFNC, 2011). Out of all four locations, Grand River not only attracted the largest population at the time of the experiment, but also received the most reactions, at 76% negative reaction rate. The number of negative reactions was counted against the total number of people that walk past the person wearing the shirt. All numbers counted were documented by a group member that is following behind the person wearing the shirt (Figure 3, Table 2).

Original Predictions

PCR

It was predicted that the forward mutant-type primer would elongate with the mutant-type DNA, as well as, the forward wild-type primer would elongate with the wild-type DNA. The reverse primer also would elongate with both the wild and mutant-type DNA. The differences between the two forward primers is a point mutation called HbS (Hemoglobin Sickle), and will be located at the last nucleotide in the forward primers, on the 3’ end, preventing elongation (Ye et al, 2001). Also, it was predicted that the control primers would annealed at 60°C, 3°C lower than the calculated melting temperature (Waterfall et al, 2001). Designed primers were predicted to anneal at 58°C, slightly lower than calculated control primers due to the number of guanines and cytosines in the primer, as well as, based on calculated annealing temperature using salt concentrations and melting temperature.

Gel Electrophoresis

           When gel electrophoresis was run with control primers with mutant DNA template, it was predicted that DNA bands would be produced at the 267 base pair mark. It was predicted that the opposite would occur when mutant control primers were ran with wild-type DNA template because the primers would have a base pair mismatch at the site of the mutation, causing no bands to be produced. This is because the control primer sequences code for the HbS mutation and, therefore, will only anneal and elongate with mutant-type DNA (Waterfall et al, 2001). Also, it was predicted that bands would be produced at 229 bp for both our designed mutant and wild-type primers. As predicted, however, mutant-type primers would only produce bands when ran against a mutant DNA template, while the same occurred when the wild-type primer was ran with wild-type DNA template. This is because the target section of DNA was 229 nucleotides in length, which when ran through a gel should produce a band at 229 bp in correspondence to the length of the target section of DNA (Waterfall et al, 2001). We analyzed gel electrophoresis by plotting the migration distance of the ladder bands on a semi log graph. Using the graph, the migration distance was overlaid showing the length of the target section DNA (Figure 2).We predicted that the migration distance would be proportional to the molecular size in base pair.

Mutagenesis

It was predicted that in part one of mutagenesis  that the best primers for both groups A and B, as well as, C and D, would yield glowing bands either in the well or at the 1000 base pair line. This was predicted based on the fact that during the first round of electrophoresis, with wild-type DNA, the mutant primers should not anneal to the DNA template and the whole DNA strand should be amplified.

The whole wild-type genome is larger than 1000 base pairs, and therefore, should not be able to move through the gel well or not at all. In part two of mutagenesis, it was predicted that the best primers would also glow in the wells for the same reasoning as in part one of mutagenesis. Overall, it was predicted that the combination of part one and two of mutagenesis would result in mutant DNA template being created (Figure 4).

Genomic Purification

It was predicted that the DNA yield of the samples would range between 3 to 8 μg, because in 1 million epithelial cells this was the theoretical range typically yielded. The typical theoretical yield helped to understand effectively how much DNA was extracted after the following purification methods.

Sickle Cell Sociology

Based on the College Foundation of North Carolina’s study on how physical appearance affects first impressions and judgments of people, it was predicted that 55% of people in a population would have a negative reaction to subjects wearing the designed “sickle cell shirt”(CFNC, 2011). It was also predicted that, versus the control person, the person wearing the designed sickle cell shirt would experience negative feelings towards being pointed out of a crowd so easily.

Continued Research

Further experiments that could be performed would be to run both the mutant-type DNA and the wild-type DNA against heterozygous DNA in a gel electrophoresis test to see whether different bands appear for each DNA type. This would allow doctors to differentiate individuals with homozygous wild-type DNA or homozygous mutant-type DNA from those with heterozygous/ homozygous sickle cell DNA. By determining the genotype of the parent at a time before a baby is produced, parents can then know if their baby is at risk for sickle cell anemia, which in turn, can help the parents plan accordingly for future pregnancies or a different birthing options. A protocol could be designed for this, in which, three different types of DNA would have to be collected: wild-type DNA, heterozygous sickle cell DNA, and homozygous sickle cell DNA. Each DNA type would have to be run against different heterozygous primers to be able to tell which DNA type is heterozygous (Waterfall et al, 2001).Weaknesses in the experimental design can include, but are not limited to: inaccurate readings of instruments, faulty instruments, and indefinite human error. All of these weaknesses could have altered experimental data, skewing hypothesis acceptance or rejection in the long run. These could be corrected by taking extra precaution in each experimental trial.

Ultimate Findings

For PCR, it was found that more experimental tests would have to be done to get the designed primers and mutagenesis to work. Due to the need for more experiments, the results in which PCR identifies and diagnoses sickle cell anemia is indeterminate. From genomic purification, the DNA yield was found to be 4 μg with a purity of 2.078. The actual frequencies of each location were: Brody at 54.8% (137/250) with the control receiving 12 reactions, Grand River at 76.5% (402/525) with the control receiving 15 reactions, Biology lecture at 18.4% (14/76) with the control receiving 2 reactions, and Organic Chemistry lecture 45.4% (193/425) with the control receiving 5 reactions. The t statistic was found to be 2.4362716 while the p-value was calculated at 0.0013.

Figure 5: Analysis of 1% TBE gel containing a GeneRuler 1 kb Plus DNA Ladder, and using an IB3-1 DNA template which was ran against control and mutant-type primers while varying annealing temperatures and PCR buffer volumes. Lane 1 contains the ladder, Lane 2 the control primers ran at an annealing temperature of 60°C, and Lanes 3-6 the designed mutant-type primers. Lanes 7 and 8 served as negative controls with Lane 7 only containing DNA and Lane 8 only containing primers. For each well, 12 μL of 6X bromophenol blue/xylene cyanol loading dye was mixed with 12 μL of either reaction cocktail (Lanes 1-6), IB3-1 DNA (Lane 7), or designed forward wild-type primers (Lane 8). Four variations of the reaction cocktails were used in the gel; two had annealing temperatures of 56°C while the others had an annealing temperature of 58°C, of those four, one at each at the different temperatures used 5 μL of 10X ThermoPol Reaction Buffer while the remaining two used 7 μL of 10X ThermoPol Reaction Buffer. Once all of the mixtures were loaded into the wells, an electric current of 100 volts was applied for about 45 minutes. As expected, no bands were found in Lane 7 due to the low quantity of DNA placed in the well. A wide band in Lane 8 was found at approximately 21 bp, caused by just the primers migrating through the gel. Lane 1 also did not show any bands, which supports the control primers, as they coded for the HbS mutation and were ran against wild-type DNA. Lanes 2-6 did not show any bands, which is consistent with the primers used; as with the control primers, the designed mutant-type primers coded for the HbS mutation. If both the control and designed mutant-type primers had been run against a mutant-type DNA template, they should have produced bands at 267 bp and 229 bp, respectively.