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(PP066) Enumeration of total and deleted mitochondria in DNA from human skin adjacent to melanomas.
Hubbard, Karen1, Pogozelski, Wendy2, Steinberg, Mark3, Orlow, Irene4, Dermody, James5, Hill, Helene6, 1 Department of Biology, New York, NY, US2 Dpartment of Chemistry, Geneseo, NY, US3 Department of Chemistry, New York, NY, US4 Epidemiology and Biostatistics Department, New York, NY, US5 Department of Microbiology and Molecular Genetics, Newark, NJ, US6 Department of Radiology, Newark, NJ, US
ABSTRACT- This research is part of a larger study to determine various damage responses in skin adjacent to excised melanomas. The real-time PCR method of Pogozelski, et al. (Mitochondrion 2: 415, 2003) employs 2 plasmids each containing amplicon inserts for, respectively, total and deleted mitochondria. The threshold cycles (Ct) for specific numbers of plasmids were compared to those of patient DNA at two or more concentrations using the same primers and probes. The total mitochondrion amplicon spans the Mitomap sequence from 1307 to 1433. The 4977 bp common deletion joins position 8483 to 13,458. Both amplicons are 127 bp and have similar melting temperatures. DNA was extracted from skin tissues obtained from wide skin excisions performed on melanoma patients at Memorial-Sloan-Kettering Cancer Center. All study participants signed informed consent and research authorization and completed an epidemiology risk factor questionaire. The Roche Light Cycler was used along with LightCycler FastStart DNA Master HybProbe Master Mix. To eliminate carry-over of amplified products, uracil replaces thymidine in the nucleotide mix and each sample contained 0.1 unit of Uracil N-glycosylase (UNG) in 10 L of reaction mixture. The samples were pre-incubated at 35o C for 25 minutes to digest carry-over amplification products and at 95o for 10 minutes to inactivate the UNG and activate the Taq polymerase before cycling began. Preliminary results indicate that deleted mitochondria range from less than 1 in 75 cells to 40 per cell. There appears to be great variation in total mitochondria which range from less than 1 per cell to greater than 5000. In the various samples, the % deleted ranges from 0.0014 to 10.6, with only 4 patients having greater than 1% deleted mitochondria per cell. The use of amplicon-containing plasmids to quantitate total mitochondria and their deletions is a powerful tool for measuring tissue damage that has not been previously exploited.
Key words: mitochondrial DNA damage, reactive oxygen species damage, real time PCR
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