Abstract
The increased knowledge of the human genome, thanks to its sequencing and to the accumulation of data that ensue from it, prompts the characterization of disease-causing genes. Especially because of the continuing expansion of cancer-related gene discovery, oncobiology is a discipline that is undergoing rapid change. Detection of point mutations in oncogenes or tumor suppressor genes associated with the multistep process of oncogenesis could be of value in patient management. These potential molecular tumor markers are essential not only for the diagnosis, prognosis, or the follow-up of the disease but also for the management of therapy (1,2). Particularly, the K-ras protooncogene is altered, by point mutations within codons estimated to be critical for the biological activity of the protein (codons 12, 13, or 61), in a wide variety of tumors (3). The incidence of these mutations can reach 95% in pancreatic carcinoma (4) and occurs in 40–60% of colorectal cancers, where they are associated with the progression from adenoma to carcinoma (5). The detection of K-ras mutations enables the understanding of cancer biology and pathogenesis with, for example, a role in the mucinous differentiation pathway (6). Alterations involving this oncogene may be of clinical importance because they can provide information for early diagnosis and clinical outcome. Their analysis within the tumor makes them of prognostic value (risk of relapse, mortality) (7). Moreover, activation of the K-ras gene has been detected not only in the tumor but also in the stools (8) of patients with colorectal cancer.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fujiyama, S., Tanaka, M., Maeda, S., et al. (2002) Tumor markers in early diagnosis, follow-up and management of patients with hepatocellular carcinoma. Oncology 62, 57–63.
Numa, F., Umayahara, K., Suehiro, Y., et al. (2001) New molecular tumor markers for endometrial cancer. Hum. Cell. 14, 272–274.
Fearon, E. R. (1993) K-ras gene mutation as a pathogenetic and diagnostic marker in human cancer. J. Natl. Cancer Inst. 15, 1978–1980.
Kondo, H., Sugano, K., Fukayama, N., et al. (1994) Detection of point mutations in the K-ras oncogene at codon 12 in pure pancreatic juice for diagnosis of pancreatic carcinoma. Cancer 73, 1589–1594.
Smith, G., Carey, F. A., Beattie, J., et al. (2002) Mutations in APC, Kirsten-ras, and p53 alternative genetic pathways to colorectal cancer. Proc. Natl. Acad. Sci. USA 99, 9433–9438.
Bazan, V., Migliavacca, M., Zanna, I., et al. (2002) Specific codon 13 K-ras mutations are predictive of clinical outcome in colorectal cancer patients, whereas codon 12 K-ras mutations are associated with mucinous histotype. Ann. Oncol. 13, 1438–1446.
Schimanski, C. C., Linnemann, U., and Berger, M. R. (1999) Sensitive detection of K-ras mutations augments diagnosis of colorectal cancer metastases in the liver. Cancer Res. 59, 5169–5175.
Sidransky, D., Tokino, T., Hamilton, S. R., et al. (1992) Identification of ras oncogene mutations in the stool of patients with curable colorectal tumors. Science 256, 102–105.
Hashimoto, T., Kobayashi, Y., Ishikawa, Y., et al. (2000) Prognostic value of genetically diagnosed lymph node micrometastasis in non-small cell lung carcinoma cases. Cancer Res. 60, 6472–6478.
Hibi, K., Robinson, C. R., Booker, S., et al. (1998) Molecular detection of genetic alterations in the serum of colorectal cancer patients. Cancer Res. 58, 1405–1407.
Bugawan, T. L., Begovich, A. B., and Erlich, H. A. (1990) Rapid HLA-DPB typing using enzymatically amplified DNA and nonradioactive sequence-specific oligonucleotide probes. Immunogenetics 32, 231–241.
Hacia, J. G. (1999) Resequencing and mutational analysis using oligonucleotide microarrays. Nature Genet. 21, 42–47.
Southern, E. M. (1996) High-density gridding: techniques and applications. Curr. Opin. Biotechnol. 7, 85–88.
Guo, Z., Guilfoyle, R. A., Thiel, A. J., et al.(1994) Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports. Nucleic Acids Res. 22, 5456–5465.
Fodor, S. P. A., Read, J. L., Pirrung, M. C., et al. (1991) Light-directed, spatially addressable parallel chemical synthesis. Science 251, 767–773.
Matson, R. S., Rampal, J., Pentoney, S. L., et al. (1995) Biopolymer synthesis on polypropylene support: oligonucleotide arrays. Anal. Biochem. 224, 110–116.
Khrapko, K. A., Lysos, Y., Khorlin, A., et al. (1989) Hybridization of oligonucleotides as a method of DNA sequencing. FEBS Lett. 256, 118–122.
Livache, T., Fouque, B., Roget, A., et al. (1998) Polypyrrole DNA chip on a silicon device: example of hepatitis C virus genotyping. Anal. Biochem. 15, 188–194.
Pease, A. C., Solas, D., Sullivan, E. J., et al. (1994) Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc. Natl. Acad. Sci. USA 24, 5022–5026.
Yershov, G., Barsky, V., Belgovskiy, A., et al. (1996) DNA analysis and diagnostics on oligonucleotide microchips. Proc. Natl. Acad. Sci. USA 14, 4913–4918.
Livache, T., Bazin, H., and Mathis, G. (1998) Conducting polymers on microelectronic devices as tools for biological analyses. Clin. Chim. Acta 278, 171–176.
Livache, T., Bazin, H., Caillat, P., et al. (1998) Electroconducting polymers for the construction of DNA or peptide arrays on silicon chips. Biosens. Bioelectron. 15, 629–634.
Livache, T., Roget, A., Dejean, E., et al. (1994) Preparation of a DNA matrix via an electrochemically directed copolymerization of pyrrole and oligonucleotides bearing a pyrrole group. Nucleic Acids Res. 11, 2915–2921.
Wu, D. Y., Ugozzoli, L., Pal, B. K., et al. (1989) Allele-specific enzymatic amplification of β-globin genomic DNA for diagnosis of sickle cell anemia. Proc. Natl. Acad. Sci. USA 86, 2757–2760.
Deng, G. A. (1988) Sensitive non-radioactive PCR-RFLP analysis for detecting point mutations at the 12th codon of oncogene c-Ha-ras in DNAs of gastric cancer. Nucleic Acids Res. 16, 6231.
Holland, P. M., Abramson, R. D., Watson, R., et al. (1991) Detection of specific polymerase chain reaction product by utilizing the 5′–3′ exonuclease activity of Thermus aquaticus DNA polymerase. Proc. Natl. Acad. Sci. USA 88, 7276–7280.
Syvanen, A. C., Aalto-Setala, K., Harju, L., et al. (1990) A primer-guided nucleotide incorporation assay in the genotyping of apolipoprotein E. Genomics 8, 684–692.
Bos, J. L., Verlaan de Vries, M., Jansen, A. M., et al. (1984) Three different mutations in the codon 61 of the human N-ras gene detected by synthetic oligonucleotide hybridization. Nucleic Acids Res. 12, 9155–9163.
Landegren, U. (1993) Ligation-based DNA diagnostics. Bioessays 15, 761–765.
Conner, B. J., Reyes, A. A., Morin, C., et al. (1983) Detection of sickle cell beta S-globin allele by hybridization with synthetic oligonucleotides. Proc. Natl. Acad. Sci. USA 80, 278–282.
Iitia, A., Mikola, M., Gregersen, N., et al. (1994) Detection of a point mutation using short oligonucleotide probes in allele-specific hybridization. Biotechniques 17, 566–573.
Lopez-Crapez, E., Chypre, C., Saavedra, J., et al. (1997) Rapid and large-scale method to detect K-ras gene mutations in tumor samples. Clin. Chem. 43, 936–942.
Nguyen, H. K., Fournier, O., Asseline, U., et al. (1999) Smoothing of the thermal stability of DNA duplexes by using modified nucleosides and chaotropic agents. Nucleic Acids Res. 27, 1492–1498.
Tombline, G., Bellizzi, D., and Sgaramella, V. (1996) Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops. Proc. Natl. Acad. Sci. USA 93, 2724–2728.
Nikiforov, T. T., Rendle, R. B., Kotewicz, M. L., et al. (1994) The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of single-stranded PCR products and their detection by solid-phase hybridization. PCR Methods Appl. 3, 285–291.
Mitsis, P. G. and Kwagh, J. G. (1999) Characterization of the interaction of lambda exonuclease with the ends of DNA. Nucleic Acids Res. 27, 3057–3063.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) In: Molecular Cloning: A Laboratory Manual, Analysis and cloning of eukaryotic genomic DNA. (Nolan, C., ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 9–16.
Caillat, P., David, D., Belleville, M., et al. (1999) Biochips on CMOS: an active matrix address array for DNA analysis. Sensors Actuators B 61, 154–162.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Lopez-Crapez, E., Livache, T., Caillat, P., Zsoldos, D. (2004). Detection of K-ras Mutations by a Microelectronic DNA Chip. In: Roulston, J.E., Bartlett, J.M.S. (eds) Molecular Diagnosis of Cancer. Methods in Molecular Medicine, vol 97. Humana Press. https://doi.org/10.1385/1-59259-760-2:337
Download citation
DOI: https://doi.org/10.1385/1-59259-760-2:337
Publisher Name: Humana Press
Print ISBN: 978-1-58829-160-8
Online ISBN: 978-1-59259-760-4
eBook Packages: Springer Protocols