An Examination of PCR methods and an Analysis of Their Success in Amplifying the TP53 Gene

By: A48614308, A45035431, and A45965216

LB 145 Cell and Molecular Biology

Tuesday 4 PM

Written by: A48614308

Revised by: A45035431

Finalized by: A45965216

Abstract

Written By: A48614308

Revised By: A45035431

Finalized By: A48614308

 The TP53 gene encodes a protein that reacts to various cell stresses and repairs mutations in the human genome known as TP53. Mutation of the TP53 gene leads to the inability for mutations to be repaired and potentially the proliferation of cancer (Petitjean et al 2007). The early diagnosis of TP53 mutation is fundamental to cancer prevention efforts. As a result, various studies have examined polymerase chain reaction (PCR) as a possible diagnosis vector for TP53 mutation. PCR is a process that can amplify segments of DNA with low efficacy in order to distinguish its presence in a genomic sample (Sherif, A.R et al 1996). The results of amplification can be examined, if their base pair lengths demonstrate any shortening or extension from the theoretical yield it would indicate mutation. PCR was employed in the reproduction of the TP53 gene in the human exon 7, which contains 110 nucleotides. The TP53 gene was amplified using primers and conditions outlined in previous studies such that a product with a length of 438 base pairs was amplified (Gessner et al 2003). It was hypothesized that if the primers listed in Gessner et al are introduced in a PCR master mix with proper conditions, then they will anneal to intronic segments preceding and following exon 7 of the TP53 gene and cause successful amplification of the target sequence with a base pair length of 438 in corresponding PCR conditions (Gessner et al 2003). We predict that a small product with 438 base pair length will be produced due to the location of the primers on the TP53 Gene (Olivier M. et al 2002). PCR amplification of the TP53 gene demonstrates the viability of PCR as a method of diagnosis for genetic diseases.

Figure 4. LB electrophoresis gel run with human DNA. a.) 1% agarose gel showing PCR results of human epithelial cell DNA with designed primers. Primers are of exon 7 from the p53 gene taken from a study done by Gessner et. al (2004). The amplified portion of DNA is shown in lanes 2, 4, and 5 in Figure 4. The band of DNA was determined to be the amplified area of DNA from the primers due to a semi-log plot test (see figure X). PCR was run by using a master mix to mix everything in. PCR cocktail contained 174 μL of nuclease-free H₂0, 30 μL 10X thermo pol buffer, 18 μL MgCl₂, 18 μL dNTP, 12 μL forward primer, 12 μL reverse primer, and 3 μL template DNA. This was then distributed between PCR tubes, each containing 50 μL of PCR cocktail. Right before putting the tubes in the thermocycler, 1 μL of Taq polymerase was added to each tube. The tubes were put in a gradient on the thermocycler, with an initial denaturing time of 5 minutes at 95°C. This was then followed by a period of 30 seconds at 95°C for denaturing, 30 seconds at 60°C for annealing (found with an equation and from Gessner et. al), and 30 seconds at 72°C for elongation. This process was repeated 35 times in the thermocycler for ideal small product production. 5μL from each PCR tube was then put into individual separate PCR tubes. 1 μL of gel loading dye (6X) was put into each of the 4 PCR tubes with 5μL, for a total of 6 μL in each tube. The mixture in each tube was then loaded into gel wells 2, 3, 4, and 5 respectively. The PCR mixture was run on a 1% agarose gel, made with 4 mL of LB (lithium borate) buffer, 36mL of deionized water, and 0.4 g of agarose sugar in an Erlenmeyer flask. This was mixed and heated until the solution was clear. After it had cooled to near-room temperature, 1 μL of GloGreen dye was added to allow the DNA to be seen by ultra violet light. b.) Semi-log plot of the molecular size as an equation of migration distance with human DNA. The semi-log plot was created from the agarose gel shown in Figure 5. Because no bands have been observed, the semi-log plot is the equation of the ladder seen in the gel. The migration distance was determined by measuring the distance of the bands from the well. The molecular size was then found by measuring the bands from the ladder and comparing the distance with another 100 base pair ladder. Based on the data points observed, the equation y =23.734x^-4.882 with an R² value of 0.97283 was found. Based on this equation it was determined that the band lengths from the gel have a molecular size of 440 base pairs in length.

Discussion

Written by: A45035431

Revised by: A48614308

Finalized by: A45965216

Cancer is one of the most widely recognized illnesses of this day, least of which because of the symptoms it engenders, rather because its ramifications extend past the illness into the family (Baider et al 1996). When missense mutations render the TP53 gene incapable of generating the p53 protein, cancer proliferation can be a result because of the diminished capacity for cells to repair themselves (Lakin, et al 1999). The TP53 mutation can be seen on multiple exons of chromosome 17 including exons 5, 6, 7, and 8.  The TP53 mutation is a result of a single base pair substitution, often A to T (Petitjean et al 2007). The correlation between TP53 mutation and the propagation of cancer has been thoroughly investigated, with multiple studies outlining ways to pre-diagnose TP53 mutation utilizing PCR (Gessner et al 2003). We hypothesized that designed primers and optimal annealing temperatures would amplify the TP53 mutation on exon 7 of chromosome 17.

Original Predictions

The amplification of the homo sapien TP53 gene on exon 7 utilizing the designed primers and conditions was observed using gel electrophoresis to reveal the length and presence of product. It was hypothesized that the forward primer would anneal at base pair 13244 until base pair 13259, which is slightly before exon 7 of the TP53 gene. The reverse primer was hypothesized to anneal at base pair 13662 until base pair 13682, which can be found slightly after exon 7 (Gessner et. al 2003).  With successful annealing, it was hypothesized that the product would be 438 base pairs long, which would be observed using gel electrophoresis in between the 400 and 500 base pair bands of a 100 base pair ladder for contrast.

Ultimate Findings

Optimal PCR conditions were found by control experiments using the lambda virus and multiple trials of PCR with human DNA. The lambda virus was used to determine the correct master mix for a PCR cocktail. It was found using a master mix of the materials worked best; all the materials were added together into a 1.5mL tube. Multiple master mixes were created determining the optimal concentration of MgCl₂, annealing temperature, and buffer. The optimal concentration of MgCl₂ was found by troubleshooting and observing bands with different concentrations of MgCl₂. Based on bands observed in gel electrophoresis, 18µL was the optimal concentration of MgCl₂. The optimal annealing temperature for human DNA were given in the published primers article and was also determined by primer melting temperatures along with an equation to find annealing temperatures (see methods). However, more troubleshooting was performed to find the optimal annealing temperature for the lambda virus and human DNA. It was found that 60˚C was the optimal annealing temperature for human DNA, while the lambda virus had an optimal annealing temperature of 54˚C. No bands were yielded after performing PCR until gel 11. In order to find the difference in master mix of gel 10 and gel 11, troubleshooting was performed on the type of buffer used. It was found that thermo pol buffer yielded better results than HlFl high fidelity buffer.(Figure 3). This may have been a result of the thermo pol buffer already containing a concentration of MgCl₂ that could work with the added MgCl₂ to aid the taq polymerase as a co-factor facilitating taq polymerase’s function.

After troubleshooting, PCR was performed using 18µL MgCl₂, an annealing temperature of 60˚C, and thermo pol buffer. This master mix yielded the best bands in gel electrophoresis. With this master mix, the designed primers were able to anneal with human DNA to amplify the TP53 gene on exon 7 (Figure 5). Figure 5 demonstrates the small product generated from PCR amplification of human DNA in-between 400 and 500 base pairs on the 100 bp ladder. Mathematical analysis of a semi log plot of this figure reveals that the band created appears where 430 base pair products should be. This result is less than 2% off the theoretical band migration length, demonstrating that the original hypothesis, that published primers and protocols would successfully amplify a desired portion of DNA was correct.

Future Implications

Written by: 48614308

The ability for small product length to be evaluated was demonstrated to be a viable means of examining the wild type variants of certain genes. Diagnosis of TP53 mutation was explored in Gessner et al where epithelial tissue from breath condensate was collected and run through a DNA capture column. Because mutations on the selected exons influenced the base pair length of the small products yielded, PCR was found to be a successful method of assaying DNA for mutations. Utilizing theoretical yields, and wild type controls, PCR is a demonstrably useful process for the isolation and diagnosis of genetic mutation (Gessner et al 2003).

Correlations between stress and the incidence of mutation have revealed that people who suffer from stress, specifically unexpressed feelings of anger and frustration correlate significantly to increased rates of cancer (Greer et al 1975). Due to the ability of PCR to examine determine discrepancies between wild type, and mutant genes, a protocol can be developed to target individuals who are determined to have abnormally high levels of stress for PCR testing to determine whether or not they may be at risk for developing cancer. PCR can be run on multiple genes that act as a bulwark for these individuals against a confirmed wild type variant, and should variations arise, health care professionals would be able to take preventative measures to prevent cancer.

Work Cited

Written By:A45035431

Finalized by: A45965216

Belyi, V. A., P. Ak, E. Markert, H. Wang, W. Hu, A. Puzio-Kuter, and A. J. Levine. 2010. The Origins and Evolution of the Tp53 Family of Genes, Cold Spring Harb Perspect Biol, 2(6): 1-18

Coates, A., S. Abraham, S. B. Kaye, T. Sowerbutts, C. Frewin, R. M. Fox, and M. H. N. Tattersall. 1983. On the receiving end—patient perception of the side-effects of cancer          chemotherapy. European Journal of Cancer and Clinical Oncology. 19: 203-208

Gessner, C., H. Kuhn, K. Toepfer, S. Hammerschmidt, J. Schauer, and H. Wirtz. 2003. Detection of p53 gene mutations in exhaled breath condensate of non-small cell lung cancer patients. Lung Cancer 43: 215-222

Greer, S. and T. Morris. 1974. Psychological attributes Of Women Who Develop Breast Cancer: A Controlled Study. Journal of Psychosomatic Research 19: 147-153

Lu-Yao, G.L, AL. Potosky, PC. Albertsen, JH. Wasson, MJ. Barry, and JE. Wennberg. Follow up Prostate Cancer Treatments after radical prostatectomy: A population-Based Study. Journal of the National Cancer Institute. 88(3-4): 166-173.

Kang, J. M, N. M. Iovine, and M. Blaser. 2006. A paradigm for direct stress-induced mutationin prokaryotes. The FASEB Journal. 14: 2475-2485

Kalendar, R., D. Lee, and A. Schulman. 2011. Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Genomics. 98: 137-144

Lakin, N. D., and S. P. Jackson. 1999. Regulation of P53 in response to DNA damage. Oncogene. 18: 7644-7655tory of the p53 tumor suppressor gene family. Dev Genes Ev. 217: 801–806

Petitjean, A., E. Mathe, S. Kato, C. Ishioka, S. V. Tavtigian, P. Hainaut, and M. Olivier. 2007. Impact of Mutant p53 Functional Properties on TP53 Mutation Patterns and Tumor Phenotype: Lessons from Recent Developments in the IARC TP53 Database, HUMAN MUTATION 28: 622-629

Schnurr, P and B, Greene, 2004, Trauma and Health: Physical health consequences of exposure to extreme stress. American Psychological Association, xii 311 pp

Sommer, R., and D. Tautz. 1989. Minimal homology requirements for PCR primers. Nucleic Acids Research 17

Barned, W,M. 1991. The fidelity of Taq ing PCR is improved by and N-terminal deletion. Gene. 112:29-35

Gunson, R.N, J. Miller, A. Leonard, and WF. Carman. 2003. Importance of PCR in the diagnosis and understanding of rotavirus illness in the community. Communicable Disease and Public Health. 6(1):63-65

WU, D.Y., and WALLACE, R.B. (1989). The ligation amplification reaction (LAR) —Amplification of specific DNA sequences using sequential rounds of template-dependent ligation. Genomics 4, 560-569.