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All PCR reactions were performed in 25 μl with an initial denaturation of 2 min at 94°C, 35 cycles of 45 s denaturation at 94°C, 45 s annealing at 60°C, and 45 s extension at 72°C. Final extension (7 min) was performed at 72°C. All the reactions were performed with Taq polymerase (Boehringer Mannheim), and the salt conditions were optimized for each primer pair with the Invitrogen PCR optimizer kit. Competitor concentrations were determined by reading the absorbency of the cloned fragments at 260 nm. Because the transferred IR was inserted in simian cells, all the primer pairs were assayed for selective amplification of human, but not simian, DNA before use in this assay. PCR reactions with primers only, or with genomic or competitor DNA with no primers, yielded no product.
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note
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We thank E. Epner and M. Groudine for advice regarding the globin locus and for globin sequences and probes; S. Strehl and M. Lalande for sharing the sequence of mitochondrial primers; S. O'Gorman for advice and site-specific recombination reagents; J. Hamlin and M. DePamphilis for sharing data before publication and for insightful comments; J. A. Huberman, M. Mechali, S. Menut, R. Gellibolian, and F. E. Indig for comments on the manuscript; and L. Brody, A. Telling, C. Navarro, and S. Wilson for technical assistance. This work was supported by grants from the NIH (CA48405, GM51104) and the G. Harold and Leila Y. Mathers Charitable Foundation. M.I.A. was supported by a postdoctoral fellowship from the Human Frontiers Science Project Organization and by a special fellowship from the Leukemia Society of America.
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