PCR and Gel Analysis of the Human SMN1 Gene Using Control Primers on IB3-1 Bronchial Cells to Diagnose SMA

Authors: Rajvinder Singh, Chris Loo, Tyler Dyke, and Kevin Thomas

Abstract

Spinal Muscular Atrophy (SMA) type 1 is an autosomal recessive disease resulting in childhood fatalities as a consequence of defective motor neuron nerve cells and overall muscle weakness of the entirety of the body (Yu-Jin et al., 2012). The Arg288Met mutation substitutes a thymine, replacing a guanine at amino acid position c.863 causing Exon 7 of the SMN1 gene to be omitted (Yu-Jin et al., 2012). We hypothesized that using experimentally determined conditions of PCR and gel electrophoresis, and completed control experiments, our designed mutant seeking primers would amplify a 460 base pair fragment of the SMN1 gene (Yu-Jin et al., 2012). PCR can also be used to determine genotype of a person’s SMN1 alleles, notifying them if they are a carrier and the chance that their newborns will have the expression of the disease. Our control primers bound to the mutant human DNA and yielded a visible band at 341 and 395 base pairs in the agarose gel allowing us to amplify a fragment of the SMN1 gene. Using PCR and gel electrophoresis with the control primers and wild type human DNA yielded visible bands at 341 and 395 base pairs. Using these methods, an effective procedure for diagnosing SMA could be developed and utilized to detect SMA at an early age to slow its fatal progression. Complementing the PCR aspect of the study is a 30 Days experiment “Simulating Spinal Muscular Atrophy”, in which we gained personal experience with the disease. We found that public reactions consisted of mostly negative facial responses and the wheelchair gave us limited mobility and access. In the psychological portion, we documented how we felt before and during the Simulating SMA events. Collectively we found that there was an instantaneous conscientious feeling as soon as we entered the SMA state of being, and from that short experience we became emotionally connected with spinal muscular atrophy.

Figure 3: Amplifying a DNA fragment of the SMN1 gene using PCR and gel electrophoresis while varying PCR reaction component volumes and annealing temperatures. The control primer used in this gel produced a band of 395 bp with a wt human DNA template (Yu-Jin et al., 2012). Running primer BLAST with these control primers confirmed they amplify two fragments of 395bp and 341bp. In the 1% agarose gel, 9μl of an Invitrogen 1Kb+ ladder was loaded into two wells. The remaining wells were loaded with 9μl as follows: PCR product type I was loaded into wells 1, 5, and 9, type II was loaded into wells 2, 6, and 10, type III was loaded into wells 3, 7, and 11, type IV was loaded into wells 4, 8, and 12. Type I included 35μl of nuclease free water, 7μl 10X Taq buffer, 2μl of Control F primer, Control R primer, Taq polymerase, and 1μl of dNTP’s and human genomic DNA template. Type II included 32μl of nuclease free water, 10μl 10X Taq buffer, 2μl of Control F primer, Control R primer, Taq polymerase, and 1μl of dNTP’s and human genomic DNA template. Type III included 40μl of nuclease free water, 5μl 10X Taq buffer, and 1μl of Control F primer, Control R primer, dNTP’s, human genomic DNA template, and Taq polymerase, respectively. Type IV included 37μl of nuclease free water, 7μl 10X Taq buffer, 2μl Taq polymerase, and 1μl of Control F primer, Control R primer, dNTP’s, and human genomic DNA template. A gradient of annealing temperatures was set increasing the chance of successful amplification. Each PCR tube cycled 30 times, denaturing at 95ºC for 50s, annealing for 1 min, and elongating at 72ºC for 1 min. Distinct annealing temperatures are listed above each well. Successful amplification of the 395bp and 341bp fragments of the SMN1 gene is seen multiple times in the gel, occurring with different PCR product types and annealing temperatures.

Discussion

Spinal Muscular Atrophy type 1 is an autosomal recessive disease that affects the alpha motor neurons concentrated highly in the spinal cord and lower brain stem (Yu-Jin et al., 2012). SMA is a common neuromuscular disease that occurs amongst newborns (Yu-Jin et al., 2012). Without the survival motor neuron proteins translated by the SMN1 gene, motor neurons in the spinal cord and muscle cells don’t function properly and therefore atrophy producing symptoms of severe muscle weakness (Leshner, 2012). The primary role of SMN proteins is the maintenance and growth of motor neurons’ dendrites and axons which fire action potential impulses leading to muscle contractions (Lefebvre et al., 1997). In particular SMA type I or Werdnig-Hoffman disease patients cannot sit, walk, and struggle to remain upright (Wirth, 2000). The Arg288Met missense mutation is one of six currently known subtle mutations causing SMA, in particular it prevents the transcription of Exon 7 on the SMN1 gene resulting in the phenotypic expression of Spinal Muscular Atrophy type 1 (Maiti et al., 2012).

The particular mutation studied is a single point mutation where Guanine mutates to Thymine on amino acid 863 on the SMN1 gene located on chromosome 5ql3 (Yu-Jin et al., 2012). We hypothesized that using experimentally determined conditions of PCR and gel electrophoresis, and completed control experiments, our designed mutant seeking primers would amplify a 460 base pair fragment of the SMN1 gene (Yu-Jin et al., 2012). In addition to running PCR and gel electrophoresis analysis on the wild type and mutant DNA, we conducted a sociological and psychological Simulating Spinal Muscular Atrophy 30 days experiment in a wheelchair and observed the reactions of strangers along with the time it took to accomplish some everyday tasks. This data was observed and recorded at Brody Cafeteria, in Holmes hall, and at Meijer shopping center. One member of the group would be wheelchair bound with a pusher as the other two recorded and observed the situation. By selecting these three locations, we targeted varying acts of kindness and negative facial reactions from people of differing age cohorts: peers and middle aged. Our feelings toward people’s responses would tell us only part of the picture but with measured activity time in completing simple, everyday tasks, we gauged what it would be like to manage the inconveniences of Spinal Muscular Atrophy.

Original Predictions

Amplifying DNA strands via allele specific PCR would indicate the presence or absence of the Arg288Met mutation. A set of primers were created using the BLAST primer database for both the mutant and wild-type DNA. These primers were intended to distinguish between the wild-type and mutant SMN1 genes based on the known base change the Arg288Met mutation incites. Prior to using the designed primers, control primers on the SMN1 gene were utilized to model expectations of a publication quality gel. Control primers will yield a visible band of 395 base pairs in the agarose because this target region of the primers has been confirmed in published SMN1 research (Yu-Jin et al., 2012). The 395 base pair fragment was successfully amplified. Our designed forward mutation primer began with base pair 980 and was designed to bind to the known mutation in mutant human DNA near the 3’ end - the reverse primer ended with base pair 1440. Our mutant seeking primers will bind to the mutant human dna and yield a visible band at 460 base pairs in the agarose allowing us to confirm the presence of the missense mutation, supporting our research hypothesis. In the 30 days experiment, the original prediction is supported by results from Brody cafe that we expect to see negative facial responses from around 60% of observed peers. These negative reactions will outnumber positive acts of kindness from our peers which we expect to be around 10%.

Results and Ultimate Findings

In order to determine optimal PCR conditions, multiple experimental trials were performed and adjusted for the concentration of DNA and primer concentration. The forward mutant primer melting point was calculated to be 58°C, the reverse mutant primer melting point was 63°C, leading us to select 54° as the proper annealing temperature. Control primers from an SMN1 gene research article were used with the purified human genomic DNA template in PCR to amplify a 395bp segment of the SNM1 gene (Yu-Jin et al., 2012). BLAST primer database determined the primers can anneal to two areas on the SMN1 gene. The 341bp and 395bp fragments were successfully amplified multiple times. Collecting data for the 30 days experiment began with a visit to Brody on Sunday 2/10. In the cafeteria we specifically measured the number of acts of kindness (people making room for us, smiling, directing us to the elevator, or holding doors) and negative facial reactions (which include staring, pointing, laughing, purposefully looking away, or gossip) from our peers in response to our wheelchair simulation of SMA type 1. In observing an approximate total of 220 people, we determined there were 105 negative facial reactions from peers and 12 peer acts of kindness toward us. In a second trial at Brody cafe, we observed 120 total peers, recorded 46 negative reactions (38.3%) and 11 acts of kindness (9.2%). Using this data, we determined expected frequencies of these reactions for future simulation events. In another sociological trial at Meijer, we observed 89 middle aged persons, recorded 27 negative reactions (30.3%) and 10 acts of kindness (11.2%). We also tested our psychological aspects of spinal muscular atrophy at Brody and Meijer. Most of our descriptions of how we felt were uncomfortable because in a wheelchair we had so much unwanted attention in these public settings.

Future Direction

The designed mutant seeking forward primer, began with base pair 980 and was 23 bases in length. The reverse primer was 23 bases in length as well, ending with base pair 1440. The DNA sequence for the forward primer was 3’-A A C A C A T C T A A G G A A A A G G A A G T T-5’ with the mutation located on the fifth base pair, Adenine, and the reverse primer was 5’-C T C T G T G G A C A C C A G T T A A A C T T-3’. The forward primer is the mutant seeking primer, and should bind to the sequence: 5’-C T T T T G G A T T C C T T T T C C T T C A A-3’ if the mutation is present. The forward, mutant seeking primer is written 5’ to 3’ and the reverse primer is written 3’ to 5’. We were unsuccessful in obtaining a mutant SMN1 gene DNA template, and would need this to continue research. We predict that the designed primers would bind to mutant SMN1 gene DNA amplifying a 460 base pair fragment allowing us to diagnose SMA resulting from the Arg288Met missense mutation. If we were unable to obtain mutant SMN1 gene DNA, designed primers would need to be ordered and Mutagenesis would need to be completed to create our own mutant SMN1 gene DNA.

http://youtu.be/uwe5Qpnh-Q4

95%