Detection of Gly178Cys Mutation on the COlA1 gene in Human IB3-1 Cells using PCR

Abstract

Type I Osteogensis imperfecta (OI) is the result of genetic mutations on the COL1A1 gene. The mutation used in this experiment is Gly178Cys. In this point mutation a guanine is changed into a thymine. This alters the amino acid from a glycine to a cysteine (Valli et al. 1991). Through the techniques of PCR and gel electrophoresis, we detected the mutation to identify DNA from IB3-1 human cells as either wild type or disease affected. For a control on PCR techniques and gel reading we used the primer from a previously successful experiment. We used 5’CCT TTC TGC TCC TTT CTC CA (sense primer) and 5’AGC AAC ACA GTT ACA CAA GG (antisense primer), which code for a four bp insertion at the end of the COL1A1 gene (Kokkro 1998). A successful PCR would replicate a band that is 43 base pairs long (Dieffenbach et al, 1993). These primers replicated no DNA however PCR techniques were proven to be correct using the Lambda as a control. The designed primers were made to detect the Gly178Cys mutation from wild type human DNA. Our forward primers are: 5’ GGC CCT TGG GTG ATG 3’ (forward mutation primer) and 5’GCC CTT GGG TGC TG 3’ (forward WT primer). The reverse primer is 5’GAT TTA CTT CGG GTT GTC G 3’. Given the time constraints for this experiment these primers were never tested so no results were found. Our team conducted a 30-day experiment to experience the everyday life with OI. For three weeks two members of the group used crutches earplugs to simulate common symptoms of OI. An experiment was conducted to gauge the reactions of people to a disabled patient with OI. It was found that people sampled were more likely to open the door for OI patients than was expected based on a Texas University study (insert citation here). A scale of depression was taken at the beginning and end of every week to measure the depression

Final Discussion

Summary

The goal of our experiment was to use PCR and gel electrophoresis to isolate and identify a mutation on the COL1A1 gene from IB3 human epithelial cells that causes Osteogenesis Imperfecta, while also incorporating symptoms of OI into our own lives to explore associations between symptoms of the disease with psychological effects. Osteogenesis Imperfecta is a genetic disease that is caused by mutations occurring in the COL1A1 gene on chromosome 17 in the human genome (Steiner, 2005). An example of a mutation is Gly178Cys. In this point mutation a guanine is changed to a Thymine. The mutation causes a decrease in the functionality of pro-α(I) chains (Gajko-Galicka, 2007). These chains are necessary for synthesizing the protein collagen, responsible for strength in bones and ligaments (Korkko, 1998). Currently the most common method of diagnosing this disease is with gene sequencing. Those tests are accurate, but they are both more expensive and time consuming then PCR. Having PCR available for patients with the potential of having hereditary Osteogenesis Imperfecta could prove to be a more effective use of time and technology.

For our PCR experiment, we designed three primers. Two of the primers are forward primers and one is reverse (Figure 1). Fprimer1 amplifies wild type DNA on the COL1A1 gene while Fprimer2 amplifies DNA with the Gly178Cys mutation. Both of them are based around the 534 nucleotide where the point mutation occurs. The nucleotide is located near the 3’ end of the primer in order to prevent hairpins or for the primer to re-anneal around the mutant base pair (Wright et al., 2013). The reverse primer is located 500 base pairs away from the two forward primers.

Besides our experimentation with primer design and electrophoresis, we also explored the realities of this disease through a series of sociological experiments. We simulated the symptoms of the disease, such as bone fractures, sprains, and hearing loss, while increasing the severity of them every week for a month by adding the use of crutches and earplugs into our day-to-day routines.  In addition a psychological depression test was taken to determine whether there were any changes to the psyche.

Predictions

For the PCR itself we will have three different temperatures cycled through 40 times. We predicted for our designed primers that a successful test, with proper annealing of temperatures, would show a band 500 base pairs long, indicating the presence of an OI causing mutation. For the control primers, the best annealing temperature was determined to be 48 degrees Celsius (see methods). All primers’ melting temperatures are within two degrees of each other to ensure the optimum amount of efficiency and accuracy for the primers (Diffenbach et al, 1993). If the PCR works then the DNA fragments produced should be 430 base pairs long. In order to correctly identify the amount of DNA amplified a gel electrophoresis needs to be done. If the DNA fragments are the correct size then the band that has been amplified through PCR should be located at the same area as the band on the DNA ladder on the gel. A wild type sample of DNA with Fprimer1 should produce 500 base pairs and show a band with similar movement to the base pair ladder. The same DNA with the Fprimer2 should not produce any kind of band on the gel. DNA with the Gly178Cys mutation should produce 500 base pair DNA fragments with the Fprimer2 and no DNA fragments with the Fprimer1. A band hypothetically will be by the 500 base pair ladder band.

We predicted that there is a causation correlation between the number of times someone is injured to the severity of the symptoms as normal daily routines will become more difficult (Widmann et al, 2002). As well as that there is an association between psychological wellbeing and complications from the disease, as overall quality of lifestyle and reactions from the public change (Cole, 1993).

Results

In an attempt to get bands, the control primers were used to try to get bands around 430 base pairs, multiple trials of the PCR were run, however none were successful (Figure 4). Throughout the semester 19 different cocktails were made and were run in six different gels. Many things were done in an attempt to get bands throughout the weeks of the experiment. The annealing temperature would range from 38⁰C to 58⁰C. DNA replication was completed a second time in an effort to have more DNA for the PCR (Figure 2). A lambda cocktail was also made and tested at the same time as an OI cocktail. The lambda cocktail had an annealing temperature of 55⁰C and the OI cocktails were run between 54⁰C to 56⁰C. In the gel, lambda had a band just below 500bp and again there was no bands at 430bp were the osteogenesis imperfecta (Figure 5). This was not because of a human technique error. The lambda and control gel showed this because they were both used the same DNA. A pipette control experiment was also used to make sure that the right amounts of ingredients were going into the cocktail and gel. 100uL of water were pipetted onto a weigh-boat 50 times to weight the amount of water. This was done twice and showed that the calibrated was correct.

A final gel was run with new DNA made by Eric Gurzell, a graduate teaching assistant for Dr. Douglas Luckie, and a changed cocktail from 1uL of dNTP to 2uL. The annealing temperature of OI cocktail was run between 48⁰C and 54⁰C. However, this still yielded the same results. In the end, this was not surprising because the scientists who designed the control primers had difficulty, getting bands as well (Korkko, 1998).

The team’s 30 days experiment as designed to study the physical, emotional, and sociological impacts of OI. The experiment included 3 different treatments over the span of 4 weeks, with severity of the symptoms increasing with each week while aiming to test the sociological impacts and a correlation with depression as impacts of Osteogenesis imperfecta. Based on a previous study from the University of Texas, it was hypothesized that most people in public would assist a handicapped person in opening a door. The outcome expected was that at least 48.6% of people at 4 locations, Pizza House, MSUFCU, Holmes Hall, and the Chemistry Building at MSU would help a handicapped stranger open a door (Lu, 2010). This hypothesis was proven, with a total of 63.6% of people opening a door for one of the handicapped team members during the study (Figure 6). To further prove such hypothesis a chi square test was run between the data collected at each of the 4 locations. A p-value of .05 was calculated, which statistically proves that the data collected at each location was significant (Figure 7). The second hypothesis over the 30 days study was that the emotional well being of an individual would changed as symptoms and side effects of Osteogenesis imperfecta changed. As predicted, the limitations and obstacles that came with the addition of crutches and earplugs into everyday life greatly increased a person’s depression level. After taking the CESD-R online test at the beginning of the experiment, team member Audrey scored a 22 out of 60 and TJ scored a 24 out of 60 possible points. By the end of the experiment Audrey’s score on the test increased to a 34 and TJ’s 28, as they had to use crutches and earplugs but continue their days as normal. Alissa and Lauren were the controls whose scores remained between low between 10 and 16 throughout the experiment (Figure 8). With the increase in depression level score in Audrey and TJ, a positive correlation between depression and side effects of the disease is evident. In conclusion, our 30 days experiment was successful in proving that disabilities experienced in Osteogenesis imperfecta patients impacts the individual both sociologically and physically

Figure 4: Control primers with Lambda DNA. To ensure proper techniques, a gel was run with the Lambda DNA and the control primers at the same time. The control primers used were run through PCR using annealing temperatures 54⁰C and 56⁰C and the Lambda cocktail was run using 55⁰C as the annealing temperature. An LB gel was used and ran at 250 volts for 15 mintues. A 1kb ladder was placed in lanes 4 and 8. The control primers were injected in lanes 5 and 6, yielding no bands, and therefore no amplification of the DNA is seen in the gel. In lane 7, is an amplification of Lambda DNA showing a band of about 500 base pairs as expected.

Future Directions

Since we were unable to get our control primers to work, and only adjusted two components of our cocktail there is a number of things that could be manipulated to get PCR to work. One of the things that could be manipulated is the PCR buffer. The buffer is used in the cocktail to keep the pH at the optimum level for replication. If the pH was too low or high no replication would occur. After the pH of the cocktail was taken the buffer would be adjusted accordingly. The PCR buffer used in the mixture contained both the Mg ²⁺ and the salt KCl. Both of these are important to make PCR work correctly (Altshuler 2006). Mg²⁺ increases the annealing temperature so it makes the binding more specific. Also, an increase in KCl salt binds base pairs below 500bp better (Altshuler 2006). The next experiments would include increasing the salt content of the cocktail to make it easier to create the shorter bands, and the Mg²⁺ for better binding. After the control primers worked experimentation will begin on the primers designed by the group for the Gly178Cys mutation.

For the 30 days with OI experiment we achieved significant results. However, we failed to encompass the entirety of OI symptoms. Including the symptoms besides bone fracture and hearing loss, such as the blue tinted sclera could add more to the data found. Also instead of using data found from a Texas University study, a control could be used to find the average frequency of door holding for non-handicapped people.