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The lab has three main projects: the first involves the analysis of human p53 binding interactions to various cytoplasmic and nuclear molecules. Our goal is to further understand the role of this important protein in regulating cancer by developing assays to determine the functional levels and binding characteristics in normal and cancerous cells. Mutations in the p53 gene, often in the DNA binding region, are associated with different cancers; therefore having a quantitative assay to measure p53 binding may be useful as a diagnostic. The second project involves analyzing various proteases in plants, using two forms of green fluorescent protein (GFP) linked via a protease sensitive peptide. This should allow us to measure protease activity in intact, living plants so that we can understand how these enzymes are regulated in the organism. The third project involves DNA computing (i.e., trying to elucidate methods for convenient computations using nucleic acids). We have developed two main methods and are trying new alternatives that may be more practical for these algorithms. We are also generating computational libraries of sequences to be used as well. These approaches aim to find methods to use DNA as a computer to solve problems that are too challenging for traditional computers.
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The Technique
The technique we describe allows for the quantitative measurement of DNA binding by human p53 in a fast and sensitive format suitable for high-throughput screening. The scintillation proximity assay (SPA) offers a distinct advantage over classic DNA binding assays that use shifted mobility in a polyacrylamide gel to measure this activity. SPA uses microscopic beads embedded with chemical scintillant and coated with antibodies that can bind specific proteins. When radiolabeled DNA is bound by the specific protein, the radioactivity is brought in close proximity to the scintillant, which then produces light. This light can be detected using a scintillation counter or a charged-coupled device (CCD) camera. There is no separation step required, so real-time measurements can be obtained in a short time. SPA has also been used to measure DNA helicase, ligand binding and kinase activity. This assay described was developed to measure to p53 DNA binding activity, but can be modified to analyze any number of DNA binding proteins. We feel this DNA binding SPA will allow us to better compare the activity in a quantitative way and potentially better characterize the functional level of the p53 protein in normal and cancer cells.
Scintillation proximity assay for DNA binding by human p53, p. 303.
