Genomic Identification and Amplification of the SCGB1A1 Gene in Homo Sapien IB3-1 Cell DNA Using PCR and Gel Electrophoresis

By: Zachary Kranz, Andrea Hess, Shelby Hughey, Mohammad Islam

LB 145 Cell and Molecular Biology

Monday and Wednesday 7 PM

Anthony Watkins, Caleigh Griffin, Rajvinder Singh

4/18/2015

Abstract

The SCGB1A1 gene is associated with an anti-inflammatory protein, uteroglobin, and is known to cause asthma if mutated (Laing et al, 1998). This experiment tried to answer the question of whether it was possible to identify and distinguish variants of this gene by using polymerase chain reaction (PCR) and gel electrophoresis. The purpose for attempting to amplify the SCGB1A1 gene using published primers was to provide evidence supporting a viable method of cloning the wild-type SCGB1A1 gene in order to differentiate the wild-type from mutants of the gene. Amplification of a desired gene segment of the SCGB1A1 gene from human IB3-1 cell DNA was attempted with PCR. This experiment was designed so that the SCGB1A1 wild-type gene was be targeted by published primers that anneal at the calculated annealing temperature of 53.25 °C and replicated by Taq polymerase during PCR (Laing et al, 1998). The genomic identity of the amplified DNA was then analyzed using gel electrophoresis to identify that PCR amplification worked and that the bands contained the desired base pair length. It was hypothesized that the SCGB1A1 gene that is wild-type for uteroglobin could be identified, isolated, and amplified using PCR and gel electrophoresis because Muller-Schottle’s primers that were previously successful in amplifying the SCGB1A1 gene were being used in this PCR technique. Analysis of the SCGB1A1 gene that went through PCR amplification and gel electrophoresis suggests that PCR did not work for this gene because there were no bands present. Slight smearing was found in one gel however this may have been the result of contamination in the PCR cocktail because it was not present in subsequent gels made with similar PCR cocktails. Future experiments with a different set of primers may be required to amplify the SCGB1A1 gene. Experiments like this are important because they help other researchers differentiate between wild-type genes and mutant ones.

Discussion

Experiment Summary

Asthma is a condition in the lungs where bronchial tubes are narrower, causing shortness of breath upon contraction (Kim and Mazza, 2011). The SCGB1A1 gene, which codes for uteroglobin, is considered to be related to asthma (Choi et al, 2000). Polymorphisms of SCGB1A1, where an adenine to guanine substitution occurs at position 38, affects the production of uteroglobin, increasing susceptibility to bronchial asthma in patients (Laing et al, 1998).

The question arises, is it possible to identify and distinguish the variants of this gene that are benign from those that could cause more harm?  It was hypothesized that with PCR amplification and gel electrophoresis the wild-type SCGB1A1 gene could be isolated, identified, and analyzed because Müller-Schöttle’s primers that were previously successful in amplifying the SCGB1A1 gene are being used in our PCR techniques (Müller-Schöttle, et al., 1999). With proper methodology, published primers, and strong controls, the 4,042 base pair length gene would be amplified (Taylor, et al., 2006).

In an attempt to detect the SCGB1A1 gene, PCR was used to amplify DNA from cultured wild-type cells of cystic fibrosis patients. The cells were obtained from biomedical facilities at Michigan State University. PCR was used in an attempt to amplify the target sequence allowing a large enough sample for analysis by gel electrophoresis (Dieffenbach, 1993). A 1 Kb+ ladder was used to create a semi-log plot to determine the exact distance traveled by the bands in the gel electrophoresis. Our ultimate predictions were that the 4,042 base pair targeted sequence on the SCGB1A1 gene would be successfully amplified with the use of gel electrophoresis and PCR.

Original Predictions

Genomic Purification

It was predicted that the SCGB1A1 gene purification would yield 15-20 micrograms of DNA because that is the yield given by the manufacturers of the DNA purification mini kit when using 10⁶ cells (Phillips et. al., 2012).

PCR

For the positive control, it was predicted that successful amplification of the target sequence in the E.Coli DNA would produce segments of length 521 base pairs when using the annealing temperature of 47.75°C because the primers used were previously used in published articles and were shown to be successful (Hanych et. al., 1993). Polymerase chain reaction was used in an attempt to amplify the SCGB1A1 gene. The optimal temperature for annealing used was 53.2 °C because the optimal temperature for annealing for the forward and reverse primers was calculated to be 55.4 °C and 53.2 °C respectively, where the higher calculated temperature could potentially break down the forward primer (Dieffenbach, 1993).

Gel Electrophoresis

The correct amplification using the forward and reverse primers in the analysis of the SCGB1A1 gene was expected to show a band of 4,042 base pairs because the forward primer anneals to the 62,419,113 – 62,419,132 base pair, and the reverse primer anneals to the 62,423,136 – 62,423,155 base pair (Taylor et. al, 2006). The difference between 62,419,132 and 62,423,136 is 4,042, which is the length of the SCGB1A1 gene.

Ultimate Findings

The results from running the gel electrophoresis showed that the attempt to amplify the target sequence was unsuccessful because no bands were visible in the trials. The published primers were unable to anneal to the template strand of DNA, and there were no distinct bands present to use the semi-log plot. This smearing may have been the result of contamination in the wells. Neither trial matched the 4,042 base pair length of the targeted sequence of the SCGB1A1 gene, refuting our hypothesis. There was error associated with determining a good temperature gradient for our PCR reactions. In addition to this, there was weakness in our initial experimental design since not enough research was performed initially to ensure we had the best methods. Over time, our methodology became stronger; however, this set us back in the beginning of our research.

Genomic Purification

The average amount of purified DNA collected was 6.775mg per trial. The average absorbance of our DNA was 1.8285, which shows our DNA was not contaminated (Volker et. al., 1982). Pure DNA has an A₂₆₀/ A₂₈₀ ratio of 1.8-2.0 in 10 mM Tris-HCl (Volker et. al., 1982). If there is strong absorbance, the ratio will be smaller, indicating contaminants are present. If there is strong absorbance at 270nm-275nm, that indicates there may be phenol groups present (Volker et. al., 1982)

PCR

It was determined that the primers were ineffective since there were multiple trials run and still there was no success in annealing. This means more experimental trials will have to be done with different designed or published primers for the SCGB1A1 gene.

Gel Electrophoresis

The E.Coli primers were used for the positive and negative controls (Hanych et. al., 1993).  For the negative control, there is primer dimer at the bottom of the gel around base pair length 20, which is expected since there was no DNA used in the PCR cocktail for the negative control. There was success in amplifying the 16S rDNA E.Coli gene, which is supported with a semi-log plot using a 1 Kb+ ladder.

Results and Ultimate Findings

PCR and gel electrophoresis methods in an attempt to amplify the SCGB1A1 gene failed. There are multiple potential explanations for this. The primers used could not anneal to the target sequence or there may have been a contamination. Another explanation is that the calculated annealing temperature might not optimal for both primers to anneal to the target sequence, requiring a temperature between the original two temperatures calculated. The annealing temperatures were calculated using the following equation: Tm = 64.9°C + 41°C (# of G’s + # of C’s in primer -16.4)/N, where N is the number of base pairs in the primers (Ingert, et al.). Instead of this equation, finding a different one that is more accurate would potentially fix that problem.

It is difficult to draw conclusions from the previous results. The results do not support nor refute the hypothesis, instead the research is not yet completed. The hypothesis is somewhat negated since the previously published primers were unsuccessful, which was not expected. A negative result was obtained, and due to this, future research is still necessary.

Figure 7. Bands developed after running a gel electrophoresis of IB3-1 cells for SCGB1A1 gene. Gel electrophoresis was conducted after PCR amplification of the SCGB1A1 gene. A PCR cocktail was made that contained 1 µl of Taq polymerase, 5 µl of PCR buffer, 3 µl of 50 mM MgSO4, 1 µl of dNTPs, 2.5 µl of each primer, 1.5 µl of template DNA, and 33.5 µl of H2O. The cocktail was run in a thermocycler at the following conditions: 5-minute initial denaturation at 95°C, then 35 cycles of: 30 seconds denature at 95°C, then 30 seconds of annealing at a gradient of 50-56°C, then 30 seconds of extension at 72°C. After these 35 cycles, there was a 5-minute final elongation phase at 72°C. A 0.8% agarose gel was made using 0.8g agarose, 95 mL distilled water, 5 mL LB Buffer and 1 µl GloGreen. 1 µl Kb+ ladder, 1µl purple (6X) no SDS loading dye and 4 µl H2O was mixed using a Vortex machine. 10 µl PCR cocktail and 5 µl of purple (6X) no SDS loading dye were mixed together with a Vortex machine as well. Wells 2-8 were loaded with 10 µl of the PCR and dye mixture. The resulting lines of bands (shown by the arrow) suggest that either the DNA was not purified enough or the primers annealed to themselves. (Narita et. al., 2002).