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Does TPEOB Interact with DNA?

Explore how TPEOB affects DNA by studying its impact on genomic and plasmid DNA using spectrophotometry and gel electrophoresis. Results indicate potential destabilizing effects on the DNA molecule. Discover more about TPEOB’s behavior for future biotechnology applications.

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Does TPEOB Interact with DNA?

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  1. Does TPEOB Interact with DNA? Scituate High School Rebecca Antonelli, Marissa Marzano, Mallory Mouradjian, Leah Staniorski

  2. TPEOB (4 ethoxybenzyl) triphenylphosphonium bromide • TPEOB is a molecule containing 4 benzene rings, 3 on one side, bound to a phosphorus ion. On the other side there is one carbon atom (CH2), the 4th benzene, then an oxygen atom, and 2 more carbon atoms (CH2, and CH3). TPEOB’s molecular formula is: C2H5OC6H4CH2P(C6H5)3Br • Molecular Weight = 477.39 AMU • Melting Point = 214°C Spooling a DNA sample Due to the central Phosphorus atom being positive, no hydrogen atoms are attached to that particular atom. Carbon (CH2)

  3. Genomic DNA Melting Using 1 μl of TPEOB and the CARY-Bio-100 Spectrophotometer, one can conclude that our molecule of choice had no affect on the native salmon DNA. The melting point stayed at 72.5°C whether TPEOB was added or not.

  4. Genomic DNA Melting (continued) While using the SmartSpec™ Plus Spectrophotometer we found that when 5 μl of our dopant was added to the cuvette containing salmon DNA, the melting point shifted. The melting point was 46.5 °C with the dopant. The control had a melting point of 68.5°C.

  5. Plasmid DNA Melting We ruled this set of data inconclusive because we did not see the absorbance level off within the temperature range we were able to test. Without this we cannot say that the DNA had melted fully. Therefore we did not plot ΔA/ΔT vs. Average Temperature to observe a shift in the melting point.

  6. Gel Electrophoresis 1 2 3 4 5 Lane 1- 4.35 mM TPEOB Lane 2- 8.7 mM TPEOB Lane 3- 17.4 mM TPEOB Lane 4- cut plasmid only (control) Lane 5- 1 kb ladder Lanes 1-4 each contained 0.37 μg of DNA. 10.0 kb 8.0 kb 6.0 kb By looking at the ladder, you can estimate that the mass of the cut plasmid is between 5 kb and 6 kb since it falls between those two points. The actual mass of cut plasmid is 6.3 kb. 5.0 kb 4.0 kb 3.0 kb 2.0 kb 1.5 kb

  7. HyperChem First we created a random DNA molecule 12 base pairs long. The order was CTTAGTACTGTG. Then we merged our TPEOB molecule with the DNA. Energy before Geometry Optimization- 5672.38

  8. HyperChem (continued) After we inserted the TPEOB molecule into the DNA molecule, we used the geometry optimization feature and the two strands of the DNA separated suggesting that the DNA had melted. Energy after optimization- 345.03

  9. Conclusions UV Spectrophotometer - When adding 1 μl of TPEOB to salmon DNA, we can conclude that it had no effect on the melting point. When adding the same amount of TPEOB to cut plasmid DNA we found our data to be inconclusive. Smart Spec Plus Spectrophotometer- When adding 5 μl of TPEOB to salmon DNA our results show that TPEOB had an effect on DNA. The salmon DNA with the TPEOB added had a lower melting point. Gel Electrophoresis- The cut plasmid DNA with 4.35 mM of TPEOB traveled the same distance as the un-doped cut plasmid. At the higher concentrations (8.7 mM, 17.4mM) the TPEOB bound to the DNA and did not permit it to travel through the gel pores. HyperChem- From the tests conducted on HyperChem we can predict that TPEOB effects the DNA molecule by destabilizing the double stranded DNA which may allow the DNA to denature at a lower temperature.

  10. Future Studies • The results that we accumulated suggest that TPEOB has a destabilizing effect on DNA. This warrants further experimentation. Possibilities include: • Bioassay for cytotoxicity • X-ray crystallography

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