Facile detection of mitochondrial DNA mutations in tumors and bodily fluids

Science

Volumn 287, Issue 5460, 2000, Pages 2017-2019

  Fliss, Makiko S ^a   Usadel, Henning ^a   Caballero, Otàvia L ^a   Wu, Li ^a   Buta, Martin R ^a   Eleff, Scott M ^a   Jen, Jin ^a   Sidransky, David ^a  

^a JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

MITOCHONDRIAL DNA; PROTEIN P53;

AMINO ACID SUBSTITUTION; ARTICLE; BLADDER TUMOR; CLINICAL ARTICLE; GENE EXPRESSION; GENE MUTATION; GENETIC MARKER; GENOME; HEAD AND NECK TUMOR; HUMAN; LUNG TUMOR; MALIGNANT TRANSFORMATION; MITOCHONDRION; POLYMERASE CHAIN REACTION; PRIORITY JOURNAL; TUMOR DIAGNOSIS; TUMOR SUPPRESSOR GENE;

AMINO ACID SUBSTITUTION; BODY FLUIDS; BRONCHOALVEOLAR LAVAGE FLUID; DNA, MITOCHONDRIAL; DNA, NEOPLASM; GENES, P53; HEAD AND NECK NEOPLASMS; HUMANS; LUNG NEOPLASMS; MUTATION; NEOPLASMS; POINT MUTATION; POLYMERASE CHAIN REACTION; POLYMORPHISM, GENETIC; SALIVA; SEQUENCE DELETION; URINARY BLADDER NEOPLASMS;

EID: 0039250954     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.287.5460.2017     Document Type: Article

References (24)

1. R. N. Lightowlers, P. F. Chinnery, D. M. Turnbull, N. Howell, Trends Genet. 13, 450 (1997).
2. MITOMAP: A Human Mitochondrial Genome Database, Center for Molecular Medicine, Emory University, Atlanta, CA (www.gen.emory.edu/mitomap. html).
3. D. L Croteau and V. A. Bohr, J. Biol. Chem. 272, 25409 (1997).
4. K. Polyak et al., Nature Genet. 20, 291 (1998).
5. 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).
6. 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°C for 30 s, 64°C for 1 min, 70°C for 3 min, three cycles; 94°C for 30 s, 61°C for 1 min, 70°C for 3 min, three cycles; 94°C for 30 s, 58°C for 1 min, 70°C for 3.5 min, 15 cycles; 94°C for 30 s, 57°C for 1 min, 70°C for 3.5 min, 15 cycles; and a final extension at 70°C 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°C for 30 s, 52°C for 1 min, and 70°C for 1 min for 25 cycles).
7. Corresponding normal tissues from four patients (874, 915,1684, and 1678) were available, and DNA was extracted from paraffin samples as described previously (10).
8. R. M. Andrew et al., Nature Genet. 23, 147 (1999).
9. Supplementary material is available at Science Online (www.sciencemag.org/feature/data/1048413.shl).
10. J. Cadet M. Berger, T. Douki, J. L. Ravanat, Rev. Physiol. Biochem. Pharmacol. 131, 1 (1997).
11. J. W. Taanman, Biochim. Biophys. Acta 1410, 103 (1999).
12. S. A. Ahrendt et al., J. Natl. Cancer Inst. 91, 332 (1999).
13. 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′-GTATTTGGATGTCAGAAACACTT-3′; mtDNA: 5′-ACTTCAGGGTCATAAAGCC-3′) or MT (p53: 5′-GTATTTGGATGTCAGAAACACTT-3′; mtDNA: 5′-ACTTCAGGGCCATAAAGCC-3′) sequences, respectively. For patient 1140, oligos 5′-ACCCGCGTCCGCGCCATGGCC-3′ and 5′-ACCCGCGTCCTCGCCATGGCC-3′ were used to detect wild-type and MT sequences, respectively.
14. O. Warburg, Science 123, 309 (1956).
15. J. W. Shay and H. Werbin, Mutat. Res. 186, 149 (1987).
16. D. R. Green and J. C. Reed, Science 281, 1309 (1998).
17. D. C. Wallace, Annu. Rev. Biochem. 61, 1175 (1992).
18. _, Proc. Natl. Acad. Sci. U.S.A. 91, 8746 (1994).
19. K. Khrapko et al., Nucleic Acids Res. 27, 2434 (1999).
20. C. Richter, J. W. Park, B. N. Ames, Proc. Natl. Acad. Sci. U.S.A. 85, 6465 (1988).
21. M. Chee et al., Science 274, 610 (1996).
22. S. A. Ahrendt et al., Proc. Natl. Acad. Sci. U.S.A. 96, 7382 (1999).
23. 2, 150 mM NaCl, 1 mM Spermidine, 1 mM adenosine triphosphate (ATP), and 5 mM dithiothreitol and analyzed on denatured 12% polyacrylamide gels [J. Jen et al., Cancer Res. 54, 5523 (1994)].
24. 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 für Krebsforschung, Deutsche Krebshilfe, and Fundação de Amparo à do Estado de São Paulo, Brazil (1998/2736-2) respectively.