Identification of Wild-Type PSEN1 Gene in Homo sapien IB3 Cell Lines and Predicted Avian Homolog Through Exon Specific PCR

By: Angela Ferrara, Alana O’Mara, Samantha Leacher, and Thomas Coon

LB 145 Biology II: Cell and Molecular Biology

Tuesday and Thursday, 7:00 PM - 9:00 PM

Anthony Watkins, Jessica Goldsworthy, and Chelsey Klein

April, 2015

Abstract

The Presenilin-1 (PSEN1) gene is primarily responsible for the neuropathophysiology of Early Onset Alzheimer’s Disease (EOAD) (Sproul et al., 2014).  Our experimental purpose is to amplify and analyze segments of PSEN1 using polymerase chain reaction (PCR) and gel electrophoresis to elucidate more about the gene, thus improving future studies on the gene’s molecular nature.  PCR permits amplification of PSEN1 sequences, which were further analyzed through gel electrophoresis.  DNA was extracted and purified from Homo sapiens and Zonotrichia albicollis (white-throated sparrow).  We hypothesized that we would be able to amplify a PSEN1 sequence through PCR with specific primers by manipulating annealing temperature with a gradient, based on successful results gained from PCR testing using temperature gradients with Escherichia coli DNA as a positive control (Sybesma et al., 2001).  Also, we hypothesized that based on an 85% alignment between the nucleotide sequences in a region of the human and sparrow PSEN1 gene that a homologous relationship can be established in regards to the PSEN1 gene and confirmed using PCR.  Primers successfully used in previously published papers were used for human DNA; new primers were designed for sparrow DNA.  Through PCR we were able to amplify the PSEN1 sequence in the sparrow genome and continue to refine the human PCR procedure.  Being able to amplify the segments of the PSEN1 gene in various species demonstrates the evolutionary conservation of the PSEN1 sequence to further propel research on the structural and functional conservation of the PSEN1 protein to understand more about the activity in preventing and treating EOAD (Jacobsen, 1999; Koonin, 2005).

Discussion

Alzheimer's disease (AD) is the most common form of dementia and has a strong genetic component with up to 80% heritability (Moon et. al., 2015).  Early-Onset Alzheimer’s Disease (EOAD) is a subtype of AD, which has been hypothesized to be heritable as well (Cruts et al., 1997).  Data suggests most EOAD cases are associated with a common mutation of the PSEN1 gene, located on chromosome 14 (Campion et. al. 1999; Mullan et al., 1992; Harvey et al., 1998).  Our research question is: How can PCR be used to target PSEN1 using designed primers to copy genome sequences in wild-type IB3 human DNA and homologous wild-type white-throated sparrow DNA?  We hypothesized that we would be able to amplify a PSEN1 sequence through PCR with specific primers by manipulating annealing temperature with a gradient, based on successful results gained from PCR testing using temperature gradients with E. coli DNA as a positive control (Sybesma et al., 2001).  Also, we hypothesized that based on an 85% alignment between the nucleotide sequences in a region of the human and white-throated PSEN1 gene that a homologous relationship can be established in regards to the PSEN1 gene because of evolutionary conservation and confirmed using PCR (Koonin, 2005).  We were unable to successfully amplify the desired PSEN1 sequence in the human wild-type DNA, which resulted in primer dimer.  However, we successfully amplified the PSEN1 sequence in the white-throated sparrow, resulting in a 360bp sequence based off of the equation calculated from the regression line, which was very close to the expected 406bp product.  Continued research on the PSEN1 gene will be highly valuable because of the limited knowledge on the current mechanism of the functional gene (Kovacs et al., 1996).   

We predicted that running PCR with the human wild-type primers will produce a 468 base pair product containing exon 7 of the PSEN1 gene because the primers are designed to anneal to base pairs 56,329-56,355 and 56,777-56,797.  We predict running PCR with the primers designed for the homologous white-throated sparrow DNA will yield a 406 base pair product because the primers were designed to anneal to base pairs 15,727,238-15,727,259  and 15,727,622-15,272,643.  Human DNA from was extracted and purified from the wild-type IB3 cells following the protocol provided by the Qiagen Generation Capture Column Kit (50) Handbook (2010).  White-throated sparrow DNA was extracted and purified from a blood sample following the protocol provided by the Qiagen DNeasy Blood and Tissue Kit (Spin Column).  PCR was conducted using modified published cocktails for both samples (Li et al. 2006).  Forward and reverse primers designed by Dr. Li were used for the wild-type DNA (2006).  The white-throated sparrow primers were self designed from the homologous fragment that aligns with human exon 12 of the PSEN1 gene.  An additional sample of E. coli was used to serve as the positive control for our PCR methods.  A PCR cocktail containing all elements except for DNA served as a negative control.  Following PCR, gel electrophoresis was used to analyze the PCR product using a 1% agarose gel with 1X TBE buffer and a 1-Kb+ DNA ladder.  The migration distances of each sample were measured and plotted on the line of best fit for the semi-log of the 1-Kb+ DNA ladder.  

The E. coli sample of DNA  that served as our positive control resulted in the expected 1,504bp product, using the 8F and 1492R primers designed for the 16S rDNA gene (Sanger et al., 1983; Taylor et al., 1983).  We chose to use E. coli as our positive control because much is known about its sequence, and it is a common baseline for PCR (Sanger et al., 1982).  For our negative control our research group will run a PCR cocktail that contains all components excluding a DNA sequence to verify that our PCR reagents are not contaminated (Noordhoek et al., 1994).  

Outside of our positive and negative control we set up two treatment samples of DNA targeting the PSEN1 gene: wild-type human DNA and homologous white-throated sparrow DNA.  These experimentally distinct treatments will give our research team a more holistic and thorough knowledge about the PSEN1 gene and its homology in avian species.  We chose the white-throated sparrow blood to be our research specimen because of their high rates of homology and the fact that avian erythrocytes are nucleated unlike mature mammals (Arctander, 1988).  

Amplification of the PSEN1 sequence in the wild-type DNA was unsuccessful using published primers by Dr. Li (Li et al., 2006).  While the primers were exactly the same, the PCR protocol was not accurately replicated based off of Dr. Li’s methods because our laboratory had different equipment and PCR components.  Instead of using touch-down PCR with Gold Buffer and AmpliTaq Gold polymerase we used regular PCR cycling with MgCl₂ Buffer and Taq polymerase.  Gold Buffer contains no MgCl₂, which was added separately in Dr. Li’s protocol (2006).  AmpliTaq Gold, used by Dr. Li, is isolated from Thermus aquaticus but has a built in hot-start function and is often used to improve primer specificity and yield of the desired target DNA (Moretti et al., 1998).  AmpliTaq Gold reduces non-specific PCR products compared to Taq polymerase (Moretti et al., 1998).  Therefore, our primer dimer results could possibly be eliminated by using AmpliTaq Gold with touch-down PCR (Moretti et al., 1998; Roux, 2009).

Figure 1: Analysis of Zonotrichia albicollis DNA. A) Agarose gel electrophoresis of the amplified PSEN1 gene in white-throated sparrow DNA. This is a photograph of white-throated sparrow DNA PCR cocktail under UV light. The bromophenol blue dye migrated farthest to represent the 1-Kb+ and 100pb DNA ladders. Lane 1 contained 3 µL of bromophenol loading dye and 7 µL 1-Kb+ DNA Ladder. Lane 2 contained 3 µL of bromophenol loading dye and 2.31x10^-4 of mg sparrow DNA that had an annealing temperature of 50°C. Lanes 3 and 6 contained 3 µL of bromophenol loading dye and 2.31x10^-4 of mg sparrow DNA that had annealing temperature of 50.6°C. Lanes 4 and 7 contained 3 µL of bromophenol loading dye and 2.31x10^-4 of mg sparrow DNA that had an annealing temperature of 51.7°C. Lane 5 contained 3 µL of bromophenol loading dye and 2.31x10^-4 of mg sparrow DNA that had an annealing temperature of 53.5°C. The expected length of the product was 406 bp. B) The resulting PCR product from the sparrow DNA was determined to be about 400 bp in length. A line of best fit was determined, and the migration distance of the sparrow DNA product was measured and found to be about 3 cm. This was plotted on the line of best fit, and the corresponding y-value was found to be about 400 bp. This was confirmed through the equation for the regression line.