
	1999 Summer Scholars Program

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Cancer Screening by Detection of p53 Mutations

By Ashwin Srinivas
Mentors: Dr. W. Barnes and Dr. P. Theodorakis
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO

The focus of this project is to develop a more efficient process of screening for cancerous tissue in blood. It is known that approximately half of all tumors contain a mutated p53 gene. By scanning for mutant p53 genes within the bloodstream, one could theoretically also screen for cancer cells within the patient's body. It has also been suggested that this type of an assay could be performed on urine and stool samples in order to detect tumors within the bladder and colon, respectively.

A p53 gene expressing plasmid grown by Dr. Wayne Barnes and Dr. Paul Theodorakis was treated with restriction enzyme Mse-1 in order to linearize the target DNA, namely the p53 gene, for better yield in PCR. Special modifications were made to the PCR process itself, with special attention to pH and level of dNTPs, each of which is surprisingly important to high fidelity. For reactions employing high levels of error-correcting enzyme, the primers used could not be standard PCR primers. Instead, special phosphorthioate-containing primers, namely ThioUTR181 and Thiol50B, were used in order to overcome the problem of primer degradation at the 3' end by error checking polymerases, such as Deep Vent and Pfu. My part of this project is to test new mixtures of DNA polymerases vs. pure Pfu, which until now has been the standard enzyme used for such PCR's. Past lab tests have shown that pure Pfu is not nearly as effective at amplification as Dr. Barnes's patented enzymes, Klentaq 1 (KT1) and Klentaq Long & Accurate (KTLA) mixture. The latter is standardly a mixture of a 47:1 ratio of KT1, a modified Taq polymerase, and Deep Vent, an error-checking enzyme nearly 90% similar to Pfu.

Unlike previous experiments conducted by others in which a colony screening process was used, this project was conducted utilizing a more efficient colony selection. Under this method both the p53 PCR product and pWB371, a vector engineered by Dr. Barnes and Dr. Theodorakis that contains the ends of the p53 gene, are transformed into yeast. Within the yeast, the pWB371 vector and the p53 gene come together by genetic recombination to form a complete plasmid. Then the yeast is added to plates lacking leucine as well as to parallel plates lacking leucine and containing 5 fluoroorotic acid (5-FOA). The minus-leucine plates will yield numerous colonies of yeast containing both the wild type and the mutated p53 gene. However, on the FOA plate only those colonies containing mutated p53 will be able to grow. This is due to the fact that the wild type p53 gene activates a URA3 gene that is linked to an artificial p53-enhanced promoter, while mutant p53 will not. The URA3 gene product then converts 5-FOA into 5-fluorouracil, which is toxic to yeast. Finally, it is now possible to compare the number of colonies titrated on the plates with 5-FOA to the number of colonies on plates not containing 5-FOA This ratio will amount to the percent of p53 genes which are mutant.

Past experiments conducted by Swiss scientist Dr. Richard Iggo showed that with the use of conventional PCR, his assay for screening for p53 mutations created a 5-8% false background reading. However, with the aforementioned modifications (and several others not here described) on the assay used in this study, preliminary tests have shown that the background can be reduced to approximately 0.2%. Though this may not make a large difference when assaying the p53 status of a biopsied tumor sample, where a large proportion of the cells have the mutated gene, such a reduction in background could be extremely useful for detecting low levels of cancerous cells in the blood of a patient who has not yet been diagnosed with cancer.

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