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note
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For direct sequencing of PCR product, a single band of appropriate size was excised from EtBr-stained agarose gel under ultraviolet (UV) light. The excised band was placed into a Costar spin-x 0.22-μm cellulose acetate filter unit, frozen at -70°C for 15 min, and centrifuged (Eppendorf microfuge) for 15 min at full speed. The DNA-containing eluate was transferred to a microcenrrifuge tube, 20 μg of glycogen was added, and DNA was precipitated with 0.2 volumes of 3 M sodium acetate and 0.3 volumes of isopropanol. DNA was sedimented (microcentrifuge), washed with 70% ethanol, dried at reduced pressure, and resuspended in 20 μl of double-distilled water. The sample was sequcnced according to the Sequenasc double-stranded protocol with 7.75 μl of DNA solution and 2.5 pmol of sequencing primer.
-
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22
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85088619549
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-
note
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s allele. It is, therefore, not yet clear whether activating mutations in G protein α chains are truly dominant, or whether they require some reduction in expression of the wild-type gene to manifest their effects.
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For isolation of genomic DNA from paraffin-embedded tissue [D. K. Wright and M. M. Manos, in PCR Protocols: A Guide to Methods and Applications, M. Innis, D. Gelfand, J. Sninsky, T. White, Eds. (Academic Press, San Diego, 1990), pp. 153-158] three to five adjacent 5-μm sections were cut from paraffin blocks and mounted on glass slides. One slide was stained with hematoxylin and eosin and used as a guide to select a region on the other slides composed entirely of tumor. With a razor blade, excess paraffin and unwanted tissue were removed from the unstained slides, and the remaining tumor tissue was scraped into a sterile 1.5-ml microcentrifuge tube. To remove contaminating paraffin, the sample was incubated with 5 μl of octane (anhydrous, Aldrich) or Hemo-De (Fischer) at room temperature for 30 min with shaking. The tissue sample was ccntrifuged (5 min, 1000g), and the supernatant was discarded. The tissue sample was extracted twice with 500 μl of absolute ethanol and dried at reduced pressure. The tissue was treated with proteinase K (0.2 mg/ml) in 100 μl of digestion buffer (50 mM tris, pH 8.5, 1 mM EDTA, 0.5% Tween 20) at 37°C overnight, the digest was centrifuged to remove undigested debris, and the DNA-containing supernatant fraction was incubated at 95°C for 8 min.
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54
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84889631391
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note
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i2 codon 205, GTGGGTGGTCAGCGGTCTGA (eight mutant codons tested).
-
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55
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84889631070
-
-
note
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We thank S. Phipps (Cetus) for technical assistance, C. Johnson (Cetus) for histologic analysis of paraffin-embedded tissue, H. Bos (University of Leiden) for reading the manuscript, R. Brescia (UCSF) for review of ovarian tumors and reading the manuscript, R. L. Davis (UCSF) for review of pituitary adenoma pathological slides, C. B. Wilson (UCSF) for providing pituitary tumor specimens, S. Masters for providing ovarian tumor specimens, A. Spada (University of Milan) for providing biochemically characterized pituitary samples, D. Spasic, L. Goda, and C. Levenson (Cetus) for synthetic oligonucleotides, and M. Dallman, D. Gospodarowicz, and R., Jaffe (UCSF) for advice. Supported in part by NIH grants (H.R.B. and C.A.L.) and grants from the Austrian Fonds Zur Forderung Der Wissenschaftlichen Forschung (K.G. and H.F.).
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