Studies conducted through Leiden University have shown a positive correlation between common cancer causing agent, UV radiation, and mutations resulting in melanoma (de Guijl et al, 2001). With the increasing destruction of the UV-absorbing ozone layer and the resulting spike in melanoma cases today, an understanding of the carcinogenic effects UV radiation has on cells is important in helping increase public awareness and precautionary actions (Dinkova-Kostova, 2008),(Anthony & Ferrett, 2003).
Do It Yourself UV Analysis
The DIY experiment analyzed the intensity of UV radiation emitted by the Raytech Versalume UV lamp compared to that of natural sunlight using an Ocean Optics USB 2000 spectrometer (Figure 2). UV beads were also used to further support conclusions (Figure 3). Results revealed that while sunlight emits considerably more intense radiation overall than the UV lamp, the UV-B radiation emitted from the lamp is much more intense than that of sunlight. This is an important factor to consider because UV-B wavelengths, between 300-240 nm, are most harmful to DNA and cause the majority of mutations (de Gruijl, 2002). UV rays potentially alter nucleic acid structures causing them to become more reactive and, if not corrected by enzymes, mutagenesis can ensue (Hochberg et al, 2006). Knowing that the DNA irradiation assays performed during this research involve UV-B radiation at higher intensities than are typically be expected from natural sunlight exposure is important in considering the context of the results. Additionally, understanding that UV-B radiation is capable of inducing the specific CDKN2A mutation examined here that can cause malignant melanoma could lead to further research on preventative measures designed to target blocking wavelengths in the UV-B range. If further research shows that this mutation is fairly commonly induced by irradiation, methods to help activate corrective enzymes and fix DNA mutations could be investigated. The results of PCR on irradiated DNA must be analyzed taking the increased UV-B intensities into account.
Hypothesis and Predictions
We hypothesized that exposure to UV radiation could generate somatic mutations in the CDKN2A gene sequence the same as present in people with a history of a predisposition to malignant melanoma. We further predicted that UV radiation will be able to produce the T to C transition at base pair 298 characteristic of this predisposition (Figure 1). The CDKN2A gene is responsible for tumor suppression by producing specific proteins such as p16, which bind to G1 cyclin-dependent kinases that inactivate Rb another tumor suppressor, consequently inactivating it. This can result in uncontrolled growth and cell division, characteristic of melanoma (Dell-Torre et al, 2001). Therefore, if UV radiation can induce mutations to DNA, it is likely that it can cause this specific mutation to the CDKN2A gene, which would likely result in cancer as confirmed by Fiorgen Foundation for Pharmacogenomics in Italy (Vignoli et al, 2008).
Genomic Purification
Purifying DNA from IB3 cells proved challenging and success was only achieved after many weeks of optimizing protocols. In the first attempt to purify DNA from human cells, the initial incubation period was 30 minutes long. For subsequent protocols, this period was varied from 1 minute to 60 minutes- which was determined ideal- in order to ensure the cells fully saturated the column. After this proved ineffective, the protocol for adding DNA Elution Solution to the column was altered, changing the initial use of only 100 μL Elution Solution without transferring the column before heating in the sandbath, to adding 200 μL Elution Solution, centrifuging, then transferring the column and adding and additional 100 μL Elution Solution. The goal of this was to improve the function of the Elution Solution, which is used to wash the DNA through the column. As successful purification still did not occur, the cells were then centrifuged for three minutes at 800 rpm to begin the purification process and then 200 μL were removed from the top of the tube for insertion into the column and incubation for one minute. This was performed to guarantee cells were being inserted into the column initially and that the problem lied in unmixed sample that never made it into the column in the first place. Gel electrophoresis failed again. Successful purification was finally achieved in changing the last centrifuge step to 14,500 rpm for 30 seconds (Figure 4). It was then determined that DNA was most likely purified in the previous failed assays, the sample was just never spun with enough force to pull it from the column.
DNA Irradiation and Error Analysis
In performing PCR on the UV irradiated purified IB3 DNA, we wanted to confirm the presence of the targeted T>C mutation on base pair 298 of the CDKN2A gene, through using mutant primer 1 and reverse primer 2. Polymerase chain reaction (PCR) was used to amplify segments of DNA suspected to contain the desired mutation. DNA was obtained from IB3 cells, human bronchial epithelial cells, from the LB 145 Laboratory through the use of the Qiagen Inc. “Generation Capture Column Kit”. Three primers were specifically designed to target wildtype and mutant DNA sequences, producing an overall 815 base pair product. Primer 1 was designed using the mutated sequence, primer 2 targeted the reverse sequence, and primer 3 contained the wildtype sequence. Gel electrophoresis was used to determine if successful amplification occurred (Wittwer et al, 1993). In attempting to increase the number of copies of product, the amount of dNTPs in the PCR reaction cocktail was increased from 2.5 μL used in both the Lambda and E. Coli LB 145 Labs (Urbance & Luckie, 2009) to 3.5 μL. An increase in dNTPs would ensure that there were enough nucleotide bases for extension to occur properly during PCR. Annealing temperature was also dropped from 58°C to 50 °C. In Figure 5, the presence of roughly 3 bands in lane 5 (w+ DNA, w+ primers) around 800-1000 base pairs indicates successful amplification of product, but the faintness of the band also indicates a small yield. To optimize the yield and obtain stronger results, the number of PCR cycles was increased from 30 to 40 cycles in order to promote additional amplification. Gel electrophoresis with this new PCR treatment, proved to be ineffective and yielded no results whatsoever. This may however, have been due to the use of a differing concentration of agarose powder, resulting in a shallower gel in which lane contents flowed out of the shallow wells before electrophoresis could be completed.
Figure 6 demonstrates the nonspecific amplification as a result of further optimization efforts. To increase strength in the bands seen in Figure 5, the amount of DNA template in the PCR reaction was increased from 0.5 μL to 0.7 μL in one assay. Another treatment run simultaneously increased PCR temperature stages by thirty seconds and the final extension by two minutes to increase desired product yield. Both of these resulted in massive nonspecific amplification that could likely be attributed to the increase in DNA template used. This was confirmed when nonspecific amplification was still obtained with further assays using the increased DNA template values but returning temperature cycling stages to their normal times.
Future Directions
Further optimization involving PCR needs to be completed to obtain both strong and specific amplification of products. Figure 5 demonstrates the desired product roughly 815 base pairs in length with great fidelity, while Figure 6 represents strong products with nonspecific amplification. The reduced yield experienced in Figure 5 could have been due to an inadequate amount of DNA template or primers, insufficient PCR extension time, or magnesium chloride (MgCl2) concentration (Henegariu et al, 1997). Increasing DNA template concentration was done to improve yield, yet product streaking occurred, suggesting that a smaller increase in DNA template would be desired. The extension period was increased in one assay and nonspecific amplification resulted, although this could have been due to the increase in DNA template and therefore further assays isolating treatments need to be conducted. MgCl2 concentrations have been shown to have strong impacts on DNA amplification, as dNTPs bind this substance, with high concentrations reducing specificity and excessively low concentrations inhibiting optimal amplification and yield (Henegariu et al, 1997),(Bendall & Molloy, 1994). Also the free magnesium ions serve as necessary enzyme cofactors for Taq polymerase involved in PCR (Henegariu et al, 1997). The use of touchdown (TD) PCR could also be employed to attempt to increase specificity. This technique involves beginning PCR temperature cycling at annealing temperatures a couple of degrees higher than calculated for the melting temperatures (Tm) of the primers to favor more specific amplification of the product (Afonina et al, 1997). The annealing temperature is then lowered 1-4°C on alternating cycles until roughly 10°C below the actual Tm is reached thus increasing the yield in successive cycles (Eckert & Kunkel, 1990).
In the future, it would be wise to incorporate the use of restriction enzymes to evaluate if any mutations occurred at all upon UV irradiated DNA sequences (Kejnovsky et al, 2004). A broader range of mutations could be tested for in this way, as opposed to allele-specific PCR used previously, which tests for only the T to C transition at base pair 298. Studies conducted by the Academy of Sciences of the Czech Republic, however, indicate that UV irradiation of DNA can inhibit the ability of restriction enzymes to cleave the DNA sequence in some instances (Kejnovsky et al, 2004). Regardless, this technique would still be very useful in mass screening for mutations in future assays.
In the future, it would be wise to incorporate the use of restriction enzymes to evaluate if any mutations occurred at all upon UV irradiated DNA sequences (Kejnovsky et al, 2004). A broader range of mutations could be tested for in this way, as opposed to allele-specific PCR used previously, which tests for only the T to C transition at base pair 298. Studies conducted by the Academy of Sciences of the Czech Republic, however, indicate that UV irradiation of DNA can inhibit the ability of restriction enzymes to cleave the DNA sequence in some instances (Kejnovsky et al, 2004). Regardless, this technique would still be very useful in mass screening for mutations in future assays.
