PCR amplification of the PAH gene in cultured human cells and homologous regions of D. pulex and S. oleracea DNA

By: A46910358, A46380516, A48690151, A46709439

LB 145 Cell and Molecular Biology

Monday and Wednesday 4:00-6:00

Eric Kontowicz, Megan Kechner, and Ahmad Tahawi

April 13, 2015

Title Page Authored by: A46380516

Title Page Revised by: A46709439

Title page Finalized by: A46910358

Abstract

Authored by: A46380516

Revised by: A46709439

Finalized by: A46910358

An experiment was conducted with the purpose of amplifying a target nucleic acid sequence in exon 7 of the human PAH gene and amplifying its homolog in Daphnia pulex and Spinacia oleracea (spinach) DNA. Exon skipping can be induced by a silent G to A mutation in exon 7 of the PAH gene at base pair (bp) 957 in cDNA, resulting in the genetic disorder Phenylketonuria (Dworniczak et al. 1990). Polymerase Chain Reaction (PCR), a DNA sequencing tool, was utilized to research the question of whether DNA evolutionary relationships of the PAH gene are stronger between humans/Daphnia or humans/spinach. Primers AP333/AP237 from published research by Dworniczak and his colleagues were used to amplify exon 7 of the human PAH gene (Dworniczak et al. 1990). Through BLAST sequencing, primers SSF20/SSR23 were designed to anneal to a homologous region in Daphnia pulex. (Madden 2002). We predicted that primers AP333/AP237 would anneal to human DNA and primers SSF20/SSR23 would anneal to Daphnia and spinach DNA because of exact matches between the 3’ ends of the primers and corresponding DNA sequences (Sommer and Tautz 1989). If primers AP333/AP237 and SSF20/SSR24 annealed to their proper locations on the PAH gene or homologous regions during PCR, then gel electrophoresis could be used to visualize bands at 212bp for human, 159bp for Daphnia, and 521bp for spinach DNA (Dworniczak et al. 1990). E. coli and primers 8F/529R at ~51℃ was a positive control because a 521bp sequence indicated that Taq was properly functioning. Results indicated average DNA absorbance ratios of 1.895 for human, 1.874 for Daphnia, and 2.170 for spinach DNA. A 212bp human sequence was amplified with primers AP333/AP237 at 52.6℃ and 159bp Daphnia sequence was amplified with primers SSF20/SSR24 at 49.9℃. No amplification of a spinach DNA occurred with primers SSF20/SSR24 at 49.9℃. Amplification of the human PAH gene is significant to diagnosing patients and researching homologous regions. Homologous regions in organisms that do not display PKU symptoms could be used to improve treatments or develop a cure (Pribat et al. 2010).

A. B.

Figure 7 A: A gel containing a ladder in well 1, an E.coli PCR cocktail in well 2, Human PCR cocktails in wells 3, 4, and 7, Daphnia in wells 5 and 6, and spinach DNA in well 8.  The pictured gel was made with 0.6g of agarose and 40 ml of lithium Borate buffer. The gel mixture was heated to melt the agarose and briefly cooled before 2ul of glo-green were added. The mixture was then left to congeal for about 10 minutes between two casting gates with a comb inserted near the top of the gel. Once the gel had solidified, the casting gates and comb were removed. Buffer was added to the buffer chambers until the gel was fully submerged. Well 1 contains 10ul of a mixture containing 6ul of a 100bp ladder and 4ul of 5X LB loading dye. 5X LB loading dye (4ul) was mixed with 6ul of each PCR cocktail containing 3ul of dNTPs, 3ul of MgCl₂, 10ul of thermopol buffer, 2.5ul of primer 8F, 2.5ul of primer 529R, 1ul Taq, a swap of E.coli, and 78ul. Then, 10ul of the E.coli PCR and loading dye cocktail was added to well 2. Each PCR cocktail was exposed to an initial 95℃ for 3 min. followed by a 95℃ for 30s, then a specific annealing temperature for 30s, and finally a 72℃ for 45s.35 cycles were performed in which after elongation the thermocycler cycled to the 30s denaturation phase. E. coli in well 2 had an annealing phase of 51.9℃. In relation to the 100bp ladder, a band at about 521 bp can be visualized in the gel in well 2. The E. coli acted as a positive control.  Wells 3, 4, and 7 contain 4ul of LB loading dye mixed with 6ul of a PCR cocktail made with 3ul of dNTPs, 10ul of thermopol buffer, 0.8ul of primer AP333, 0.8ul of primer AP237, 1ul Taq, 5ul of human DNA, and 79.4ul of water. The human PCR cocktail loaded in wells 3, 4, and 7 were exposed to a annealing temperatures of 54.2℃ for well 3, 56℃ for well 4, and 51.9℃ for well 7. In relation to the 100bp ladder, a band can be visualized at about 212bp in wells 3, 4, and 7. Wells 5 and 6 contain 4ul of 5X LB loading dye mixed with 6ul of a PCR cocktail made with 3ul of dNTPs, 3ul of MgCl₂, 10ul of thermopol buffer, 0.8ul of primer SSF20, 0.8ul of primer SSR23, 1ul of Taq, 5ul of Daphnia DNA, and 76.4ul of water. The Daphnia PCR cocktail loaded in well 5 had an annealing temperature of 49.9℃.The Daphnia PCR cocktail loaded in well 6 had an annealing temperature of 48.7℃. In relation to the 100bp ladder, a very faint band at about 159bp can be visualized. Well 8 contains 4ul of 5X LB loading dye mixed with 6ul of a PCR cocktail made with 3ul of dNTPs, 3ul of MgCl₂, 10ul of thermopol buffer, 0.8ul of primer SSF20, 0.8ul of primer SSR23, 1ul of Taq, 5ul of spinach DNA, and 76.4ul of water. The spinach PCR cocktail loaded in well had an annealing temperature of 48.7℃. In relation to the research question, this image indicates successful amplification of a 212bp human DNA sequence using primers AP333 and AP237 from published research by Dworniczak and his colleagues (Dworniczak, 1990), as well as the use of E. coli as a positive control. It also shows the minimal success of primers SSF22/SSR20 to confirm homologous regions of the PAH gene in daphnia and spinach DNA.

B: A semi log plot based on data observed from a 100bp ladder. A 100bp ladder was analyzed and the distance each band traveled from the well was measured in cm. In this figure, the number of base pairs of each corresponding band was graphed against the distance the bands traveled from the wells. The result of this table is that as the number of base pairs decrease in the graph, the distance that each band travels from the wells increases. The formula seen in this table is the formula for the line of best fit on the graph, which is y=40024x^-3.006. This formula was then used to calculate the exact amount of base pairs each product contained when the distance traveled was measured. In this case the semi log plot does not provide accurate data because insufficient values are given for calculation. The coefficient of determination (R²) is 0.89452. In relation to the research question, the semi log created using the 100bp ladder can be used to determine if bands observed from the annealing of primers AP333/AP237 and SSF20/SSR23 are the desired target sequences.  

Discussion

Authored by: A46709439

Revised by: A46380516

Finalized by: A48690151

Background and Summary

Phenylketonuria is an autosomal recessive disease that is associated with a mutation within the phenylalanine hydroxylase (PAH) gene (Blau et al. 2010), Mutations within the PAH gene can lead to hyperphenylalaninemia (HPA) (Scriver 2007). More than 500 different mutations are known to exist and the majority of these mutations are point mutations (Zschocke et al. 2011). A mutation within PAH results in an inability to metabolize phenylalanine into tyrosine, thus leading to toxic amounts of phenylalanine in the blood (Blau et al. 2010). The silent mutation is a G to A substitution found in exon 7 at nucleotide 957 in the DNA sequence (Dworniczak et al. 1990). This nucleotide substitution produces (GTGGCT-> GTAGCT) an Alu I site, but does not change the amino acid synthesized, valine, also referred to as a silent mutation (Dworniczak et al. 1990). Although PKU does decrease overall IQ, it is not expected to decrease life expectancy if a strict diet is maintained and phenylalanine within the bloodstream is monitored (Blau et al. 2010). Identifying homologous regions within other organisms could help with identifying evolutionary origins of the gene. Previous studies have indicated that mutations in the PAH gene have occurred after the divergence of the Caucasoid and the Mongoloid peoples (Eisensmith et al. 1992).  The direction of our research is defined by the research question: can exon 7 of the PAH gene in humans be successfully amplified using published methods and PCR primers AP237 and AP333 from Dworniczak et al. and will designed primers SSF20 and SSR23 anneal to homologous DNA regions within other organisms under ideal conditions? If primers AP 333, AP 237, SSF20, and SSR23 anneal in their proper location of the PAH gene using PCR, then agarose gel electrophoresis will indicate a band of 212bp for human DNA and 159bp for Daphnia pulex DNA (Dworniczak et al. 1990). E. coli was used as the positive control to ensure that Taq polymerase is functioning properly and resulted in a 521bp band, while spinach served as a negative control for our designed primers. A semi log plot was created, for reference, using a 1kb+ ladder and was used to accurately define the length that each product traveled down the gel.

Original Predictions

Two primers were adapted from Dworniczak et al. the forward primer, AP 333, and the reverse primer, AP 237, used to anneal to human DNA (Dworniczak et al. 1990). Based on the figures created by Dworniczak, the product after gel electrophoresis was predicted to be between 210-213 base pairs (Dworniczak et al. 1990). Comparing the location of this band in the gel to the length it has traveled down the 1kb+ ladder gave a visual estimate as to how many base pairs the product contains. A second experiment using E. coli DNA and primers 8F and 529R, forward and reverse respectively, was performed as a control to ensure Taq polymerase is functioning properly. With the successful amplification of the E. coli DNA, gel electrophoresis will result in a 521bp product when compared to a 1kb+ ladder (Farrelley et al. 1995). For a future research perspective, a homologous region in Daphnia pulex DNA was amplified using primers we designed, SSF20 and SSR23, and a 159bp band was expected.

Homolog Experiment

While performing the PCR experiment with human DNA, a PCR experiment was performed simultaneously using DNA extracted from Daphnia pulex. Forward primer SSF20 and reverse primer SSR23 were created by analyzing the Daphnia genome using BLAST and used in a PCR cocktail. The cocktails were then analyzed using gel electrophoresis. Based on the annealing location of our primers, a 159bp sequence should be amplified and present in the agarose gel. The homologous region in the DNA of Daphnia pulex was amplified successfully using our designed primers and 159bp product was present in the gel. A separate PCR experiment was performed with spinach DNA extracted from spinach leaves to test if our primers, SSF20 and SSR23, would anneal to spinach DNA because of the similarity of the primers and the spinach DNA sequence. Gel electrophoresis was run, using spinach DNA, and a 521bp product should be visible on the gel after completion.

Results and Findings

Multiple trials were performed in order to obtain the optimal temperatures and cocktail mixtures. In addition, the amount of magnesium ions was increased by increasing the concentration of magnesium chloride (MgCl₂) in the cocktail, which can affect the DNA polymerase activity, to create the optimal annealing conditions (Ignatov et al. 2002). Through trial and error, it was found that the optimal temperature for human DNA was 54℃. Upon completion of gel electrophoresis using cocktails containing human DNA and primers AP 333 and AP 237, a product of 212bp was visible under ultraviolet light. As a control to confirm the Taq polymerase was functioning properly, PCR cocktails were made using E. coli DNA and forward primer 8F and reverse primer 529R. Multiple trials were performed to find the ideal temperature, 51℃, and cocktail mixture, which had an increased concentration of MgCl₂. Gel electrophoresis produced a band of 521bp when compared to a 1kb+ ladder. Further analysis of a 1kb+ ladder provided data used to create a semi log plot, which was used to specify the exact location and size of each product. The genome purification of human cells was performed using the Qiagen Generation Capture Column and its absorbance was tested in the Epoch microplate spectrophotometer. The resulting DNA had a protein contamination ratio (260/280 ratio) of 1.9 and resulted in 155 ng of DNA. Extraction of daphnia DNA was performed with the Qiagen DNAeasy Blood/Tissue kit and spinach DNA was extracted from spinach leaves (See DNA extraction in Methods section). Absorbance of both DNA’s was tested. The Daphnia DNA had a 260/280 ratio of 2.8 and contained 5 ng of DNA. The spinach DNA had a 260/280 ratio of 2.17 and contained 18 ng of DNA. The results of the gel electrophoresis of the human DNA and E. coli DNA both confirmed our hypothesis of a 212bp product and a 521bp product, respectively. Our genome purification of human, Daphnia and spinach DNA resulted in little protein contamination and was of sufficient quality to perform PCR.

Problems and Errors

There are multiple errors that could arise during PCR, which could negate our hypothesis. Non-functioning Taq polymerase, when the Taq polymerase fails to add the correct nucleotide bases, or incomplete denaturation, meaning the DNA has not been completely split preventing our primers from annealing correctly, could be some of the causes of failed PCR (Innis and Gelfand 1990). Our data for DNA absorbance of human DNA was optimal. Our ratio, 1.9, was close to pure DNA, 1.8, and the 155 ng was sufficient. However, the amount of spinach and Daphnia DNA was much lower, which could be the reason bands were not obtained using spinach DNA and only faint bands of Daphnia were present. The 260/280 ratio provides evidence of protein contamination in Daphnia, resulting in faint bands (Ghatak et al. 2013). The genome purification stage was repeated once for human DNA and once for Daphnia. The first collection of human DNA resulted in a 260/280 ratio of 2.6 and the first collection of Daphnia DNA produced inconclusive results, possibly caused by improper incubation times or temperatures. Over the course of many experiments, gel electrophoresis produced faint bands. The loading dye, purple 6x, was used in 1.5% agarose gels and is listed as best used for products near 300bp (Pai et al. 2002). This dye was used with cocktails containing human and E. coli DNA, producing products outside of this range, which we predict is the reason that the bands were so faint. A separate orange 5x LB loading dye was used which had an optimal product size of 50bp (Pai et al. 2002). This dye also resulted in faint bands for Daphnia, E. coli and spinach DNA. Daphnia and E. coli because our desired products were 159 and 521bp, outside of the optimal range, and no product was obtained from the spinach DNA.