{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T01:06:38Z","timestamp":1791939998076,"version":"4.3.4"},"reference-count":25,"publisher":"American Association for the Advancement of Science (AAAS)","issue":"5460","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Science"],"published-print":{"date-parts":[[2000,3,17]]},"abstract":"<jats:p>Examination of human bladder, head and neck, and lung primary tumors revealed a high frequency of mitochondrial DNA (mtDNA) mutations. The majority of these somatic mutations were homoplasmic in nature, indicating that the mutant mtDNA became dominant in tumor cells. The mutated mtDNA was readily detectable in paired bodily fluids from each type of cancer and was 19 to 220 times as abundant as mutated nuclear p53 DNA. By virtue of their clonal nature and high copy number, mitochondrial mutations may provide a powerful molecular marker for noninvasive detection of cancer.<\/jats:p>","DOI":"10.1126\/science.287.5460.2017","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T05:40:21Z","timestamp":1027748421000},"page":"2017-2019","source":"Crossref","is-referenced-by-count":661,"title":["Facile Detection of Mitochondrial DNA Mutations in Tumors and Bodily Fluids"],"prefix":"10.1126","volume":"287","author":[{"given":"Makiko S.","family":"Fliss","sequence":"first","affiliation":[{"name":"Department of Otolaryngology\u2013Head and Neck Surgery, Head and Neck Cancer Research Division,"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Henning","family":"Usadel","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology\u2013Head and Neck Surgery, Head and Neck Cancer Research Division,"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ota\u0301via L.","family":"Caballero","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology\u2013Head and Neck Surgery, Head and Neck Cancer Research Division,"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Wu","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology\u2013Head and Neck Surgery, Head and Neck Cancer Research Division,"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Martin R.","family":"Buta","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology\u2013Head and Neck Surgery, Head and Neck Cancer Research Division,"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Scott M.","family":"Eleff","sequence":"additional","affiliation":[{"name":"Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jin","family":"Jen","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology\u2013Head and Neck Surgery, Head and Neck Cancer Research Division,"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Sidransky","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology\u2013Head and Neck Surgery, Head and Neck Cancer Research Division,"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"221","reference":[{"key":"e_1_3_1_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0168-9525(97)01266-3"},{"key":"e_1_3_1_3_2","unstructured":"MITOMAP: A Human Mitochondrial Genome Database Center for Molecular Medicine Emory University Atlanta GA (www.gen.emory.edu\/mitomap.html)."},{"key":"e_1_3_1_4_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.272.41.25409"},{"key":"e_1_3_1_5_2","doi-asserted-by":"publisher","DOI":"10.1038\/3108"},{"key":"e_1_3_1_6_2","unstructured":"Paired normal and tumor specimens along with blood and bodily fluids were collected after surgical resections with prior consent from patients in the Johns Hopkins University Hospital. Tumor specimens were frozen and microdissected on a cryostat so that the tumor samples contained greater than 70% neoplastic cells. DNA from tumor sections was digested with 1% SDS\/Proteinase K extracted by phenol-chloroform and ethanol precipitated. Control DNAs from peripheral lymphocytes matched normal tissues urine saliva and BAL fluid were processed in the same manner as described in (12)."},{"key":"e_1_3_1_7_2","unstructured":"Mitochondrial DNAs were amplified with overlapping primers (4) in PCR buffer containing 6% dimethyl sulfoxide. About 5 to 20 ng of genomic DNA was subjected to the step-down PCR protocol: 94\u00b0C for 30 s 64\u00b0C for 1 min 70\u00b0C for 3 min three cycles; 94\u00b0C for 30 s 61\u00b0C for 1 min 70\u00b0C for 3 min three cycles; 94\u00b0C for 30 s 58\u00b0C for 1 min 70\u00b0C for 3.5 min 15 cycles; 94\u00b0C for 30 s 57\u00b0C for 1 min 70\u00b0C for 3.5 min 15 cycles; and a final extension at 70\u00b0C for 5 min. PCR products were gel-purified with a Qiagen gel extraction kit (Qiagen) and sequence reactions were performed with Thermosequenase (Perkin-Elmer) with the cycle conditions (95\u00b0C for 30 s 52\u00b0C for 1 min and 70\u00b0C for 1 min for 25 cycles)."},{"key":"e_1_3_1_8_2","unstructured":"Corresponding normal tissues from four patients (874 915 1684 and 1678) were available and DNA was extracted from paraffin samples as described previously (10)."},{"key":"e_1_3_1_9_2","doi-asserted-by":"publisher","DOI":"10.1038\/13779"},{"key":"e_1_3_1_10_2","unstructured":"Supplementary material is available at Science Online (www.sciencemag.org\/feature\/data\/1048413.shl)."},{"key":"e_1_3_1_11_2","first-page":"1","volume":"131","author":"Cadet J.","year":"1997","unstructured":"Cadet J., Berger M., Douki T., Ravanat J. L., Rev. Physiol. Biochem. Pharmacol. 131, 1 (1997).","journal-title":"Rev. Physiol. Biochem. Pharmacol."},{"key":"e_1_3_1_12_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0005-2728(98)00161-3"},{"key":"e_1_3_1_13_2","doi-asserted-by":"publisher","DOI":"10.1093\/jnci\/91.4.332"},{"key":"e_1_3_1_14_2","unstructured":"Subcloning of PCR fragments into phage vector was performed according to the manufacturer's instructions (Stratagene). Titered plaques were plated and subjected to hybridization with tetramethylammonium chloride as a solvent. Positive signals were confirmed by secondary screenings. Oligonucleotides (oligos) used for this assay were as follows. For patient 1113 p53 and mtDNA sequence alterations were detected with oligos containing either wild-type (p53: 5\u2032-GTATTTGGATGTCAGAAACACTT-3\u2032; mtDNA: 5\u2032-ACTTCAGGGTCATAAAGCC-3\u2032) or MT (p53: 5\u2032-GTATTTGGATGTCAGAAACACTT-3\u2032; mtDNA: 5\u2032-ACTTCAGGGCCATAAAGCC-3\u2032) sequences respectively. For patient 1140 oligos 5\u2032-ACCCGCGTCCGCGCCATGGCC-3\u2032 and 5\u2032-ACCCGCGTCCTCGCCATGGCC-3\u2032 were used to detect wild-type and MT sequences respectively."},{"key":"e_1_3_1_15_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.123.3191.309"},{"key":"e_1_3_1_16_2","doi-asserted-by":"publisher","DOI":"10.1016\/0165-1110(87)90028-5"},{"key":"e_1_3_1_17_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.281.5381.1309"},{"key":"e_1_3_1_18_2","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.bi.61.070192.005523"},{"key":"e_1_3_1_19_2","first-page":"8746","volume":"91","author":"___","year":"1994","unstructured":"___, Proc. Natl. Acad. Sci. U.S.A. 91, 8746 (1994).","journal-title":"Proc. Natl. Acad. Sci. U.S.A."},{"key":"e_1_3_1_20_2","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/27.11.2434"},{"key":"e_1_3_1_21_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.85.17.6465"},{"key":"e_1_3_1_22_2","doi-asserted-by":"publisher","DOI":"10.1126\/science.274.5287.610"},{"key":"e_1_3_1_23_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.96.13.7382"},{"key":"e_1_3_1_24_2","unstructured":"Fragments containing mutations were PCR amplified and then ethanol precipitated. For each mutation discriminating oligos that contained the mutated base at the 3\u2032 end were designed (TAACCATA-3\u2032 for patient 915 and TCTCTTACC-3\u2032 for patient 898). Immediately adjacent [ 32 P] end-labeled 3\u2032 sequences (5\u2032-CACACTACTA-3\u2032 for patient 915 and 5\u2032-TTTAACCAG-3\u2032 for patient 898) were used as substrate together with discriminating oligos for the ligation reaction. After a denaturing step of 95\u00b0C for 5\u2032 the reactions were incubated for 1 hour at 37\u00b0 in the presence of T4 DNA ligase (Life Technologies Rockville MD) in a buffer containing 50 mM tris-HCl 10 mM MgCl 2 150 mM NaCl 1 mM Spermidine 1 mM adenosine triphosphate (ATP) and 5 mM dithiothreitol and analyzed on denatured 12% polyacrylamide gels ["},{"key":"e_1_3_1_24_3","first-page":"5523","volume":"54","author":"Jen J.","year":"1994","unstructured":"Jen J., et al., Cancer Res. 54, 5523 (1994)].","journal-title":"Cancer Res."},{"key":"e_1_3_1_25_2","unstructured":"We thank K. Polyak and B. Vogelstein for technical advice and critical review of the manuscript and R. Yochem for technical assistance. Supported by NIH grants RO1 DE 012488 RO1 CA77664 PO1 CA 58184 and U01 CA 84986. H.U. and O.L.C. are supported by a grant of the Dr. Mildred Scheel-Stiftung f\u00fcr Krebsforschung Deutsche Krebshilfe and Funda\u00e7\u00e3o de Amparo \u00e0 do Estado de S\u00e3o Paulo Brazil (1998\/2736-2) respectively."}],"container-title":["Science"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.science.org\/doi\/pdf\/10.1126\/science.287.5460.2017","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,13]],"date-time":"2024-01-13T05:46:34Z","timestamp":1705124794000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.science.org\/doi\/10.1126\/science.287.5460.2017"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2000,3,17]]},"references-count":25,"journal-issue":{"issue":"5460","published-print":{"date-parts":[[2000,3,17]]}},"alternative-id":["10.1126\/science.287.5460.2017"],"URL":"https:\/\/doi.org\/10.1126\/science.287.5460.2017","relation":{},"ISSN":["0036-8075","1095-9203"],"issn-type":[{"value":"0036-8075","type":"print"},{"value":"1095-9203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2000,3,17]]}}}