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Detection of mutations in RET proto-oncogene codon 634 through double tandem hybridization

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Abstract

We developed a procedure to detect the 7 point mutations at Cys634 of the proto-oncogene RET, which is responsible for medullary thyroid carcinoma (MTC). Genomic DNA was prepared from blood samples obtained from normal and MTC-affected individuals belonging to a family with a history of the disease. The RET genotype for each individual was first established by performing restriction and sequencing analyses. Single-stranded target DNA was prepared by asymmetric polymerase chain reaction (PCR) amplification of a 93-bp fragment containing Cys634. The target was annealed with pairs of prelabeled stacking oligonucleotides designed to create appropriate 7-nucleotide gaps, which served as the sites of subsequent hybridization with glass-immobilized 7-mer probes. The target-stacking oligonucleotide duplexes were hybridized with DNA chips containing a set of eight 7-mer probes designed to detect the wild-type sequence and the seven point mutations described. We tested two sets of immobilized probes containing internal or 5′-terminal codon-634 single-base variations. Both groups of probes were able to discriminatively identify the mutations. The hybridization patterns indicated that the disease in this family was due to the C634Y mutation, in accord with the original sequence analysis. The hybridization-based mutation assignment was additionally supported by determination of the control homozygous and heterozygous hybridization patterns produced with synthetic targets having the normal or codon 634 mutant sequences. The effects of mismatch type and nearest-neighbor sequences on the occurrence of false-positive (mismatched) hybridizations are discussed.

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References

  1. Riordan, J. R., Rommens, J. M., Kerem, B., Alou, N., Rozmahel, R., Grzelczak, Z., et al. (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245, 1066–1073.

    Article  PubMed  CAS  Google Scholar 

  2. Mathew, C. G. (1999) DNA diagnostics: goals and challenges. Br. Med. Bull. 55, 325–339.

    Article  PubMed  CAS  Google Scholar 

  3. Little, D. P., Braun, A., Darnhofer-Demar, B., et al. (1997) Detection of RET proto-oncogene codon 634 mutations using mass spectrometry. J. Mol. Med. 75, 745–750.

    Article  PubMed  CAS  Google Scholar 

  4. Eng, C. (1999) RET proto-oncogene in the development of human cancer. J. Clin Oncol. 17, 380–393.

    PubMed  CAS  Google Scholar 

  5. Tsui, L. O. (1992) The spectrum of cystic fibrosis mutations. Trends Genet. 8, 392–398.

    PubMed  CAS  Google Scholar 

  6. Thompson, M. and Furtado, L. M. (1999) High density oligonucleotide and probe arrays for the analysis of target DNA. Analyst 124, 1133–1136.

    Article  PubMed  CAS  Google Scholar 

  7. Walter, G., Bussow, K., Cahill, D., Lueking, A. and Lehrach, H. (2000) Protein arrays and molecular interaction screening. Curr. Opin. Microbiol. 3, 298–302.

    Article  PubMed  CAS  Google Scholar 

  8. Celis, J., Kruhoffer, M., Gromova, I., et al. (2000) Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics. FEBS Lett. 480, 2–16.

    Article  PubMed  CAS  Google Scholar 

  9. Southern, E. M., Maskos, U. and Elder, J. K. (1992) Analyzing and comparing nucleic acid sequences by hybridization to arrays of oligonucleotides: evaluation using experimental models. Genomics 13, 1008–1017.

    Article  PubMed  CAS  Google Scholar 

  10. Beattie, K. L., Beattie, W. G., Meng, L., et al. (1995a) Hybridization of DNA targets to glass-tethered oligonucleotide probes. Molec. Biotechnol. 4, 213–225.

    CAS  Google Scholar 

  11. Yershov, G., Barsky, V., Belgovskiy, A., et al. (1996) DNA analysis and diagnostics on oligonucleotide microchips. Proc. Natl. Acad. Sci. USA 93, 4913–4918.

    Article  PubMed  CAS  Google Scholar 

  12. Maskos, U. and Southern, E. M. (1992) Parallel analysis of oligodeoxyribonucleotide (oligonucleotide) interactions. I. Analysis of factors influencing oligonucleotide duplex formation. Nucleic Acids Res. 20, 1675–1678.

    Article  PubMed  CAS  Google Scholar 

  13. Beattie, K., Beattie, W., Meng, L., et al. (1995b) Advances in genosensor research. Clin. Chem. 41, 700–706.

    PubMed  CAS  Google Scholar 

  14. Drmanac, R., Drmanac, S., Strezoska, Z., et al. (1993) DNA sequence determination by hybridization: a strategy for efficient large-scale sequencing. Science 260, 1649–1652.

    Article  PubMed  CAS  Google Scholar 

  15. Maldonado-Rodriguez, R., Espinosa-Lara, M., Calixto-Suárez, A., Beattie, W. G., and Beattie, K. L. (1999a) Hybridization of glass-tethered oligonucleotide probes to target strands prenanealed with labeled auxiliary oligonucleotides. Mol. Biotechnol. 11, 1–12.

    PubMed  CAS  Google Scholar 

  16. Maldonado-Rodriguez, R., Espinosa-Lara, M., Loyola-Abitia, P., Beattie, W. G., and Beattie, K. L. (1999b) Mutation detection by stacking hybridization on genosensor arrays. Mol. Biotechnol. 11, 13–25.

    PubMed  CAS  Google Scholar 

  17. Maldonado-Rodríguez R. and Beattie K. L. (2001a) Analysis of nucleic acids by tandem hybridization on oligonucleotide microarrays. Methods Mol. Biol. 170, 157–171.

    PubMed  Google Scholar 

  18. Maldonado-Rodriguez, R. and Beattie, K. L. (2001b) Nucleic acid analysis using sequence-targeted tandem hybridization. US Patent # 6,268,147 B1.

  19. Sherlock, G., Hernandez-Boussard, T., Kasarskis, A., et al. (2001) The Stanford Microarray Database. Nucleic Acids Res. 29, 152–155.

    Article  PubMed  CAS  Google Scholar 

  20. Matteucci, M. D. and Caruthers, M. H. (1992) Synthesis of deoxyoligonucleotides on a polymer support. Biotechnology 24, 92–98.

    PubMed  CAS  Google Scholar 

  21. Beattie, K. L. and Frost J. D. III. (1992) Porous wafer for segmented synthesis of biopolymers. US Patent # 50,175,209.

  22. Beattie, K. L., Logsdon, N. J., Anderson, R. S., et al. (1988) Gene synthesis technology: Recent developments and future prospects. Appl. Biochem. Biotechnol. 10, 510–521.

    CAS  Google Scholar 

  23. Beattie, K. L. and Hurst, G. D. (1994) Synthesis and use of oligonucleotide libraries. In: Innovation and Perspectives in Solid Phase Synthesis, Proceedings of the 34d International Symposium on Solid Phase Synthesis (Epton, R., ed.), Mayflower Worldwide Ltd., Birmingham, UK, pp. 69–76.

    Google Scholar 

  24. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Habor Laboratory Press, U.S.A.

    Google Scholar 

  25. Siegelman, M., Mohabeer, A., Fahey, T. J. III, et al. (1997) Rapid, non-radioactive screening for mutations in exons 10, 11 and 16 in the RET protooncogene associated with inherited medullary thyroid carcinoma. Clin. Chem. 43, 453–457.

    PubMed  CAS  Google Scholar 

  26. Gyllensten, U. B. and Erlich, H. A. (1988) Generation of single stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA DQα locus. Proc. Natl. Acad. Sci. USA 85, 7652–7656.

    Article  PubMed  CAS  Google Scholar 

  27. Reyes-López, M. A., Méndez-Tenorio, A., Maldonado-Rodríguez, R., et al. (2003) Fingerprinting of prokaryotic 16S rRNA genes using oligodeoxyribonucleotide microarrays and virtual hybridization. Nucl. Acids Res. 31, 779–789.

    Article  PubMed  CAS  Google Scholar 

  28. Méndez-Tenorio, A., Beattie, K. L., Doktycz, M.J., et al. (2002) Design of oligonucleotide probes for genosensor chips. Bioinformatics, submitted.

  29. Allawi, H. T. and SantaLucia, J. (1997) Thermodynamics and NMR of internal G-T mismatches in DNA. Biochemistry 36, 10581–10594.

    Article  PubMed  CAS  Google Scholar 

  30. Allawi, H. T. and SantaLucia, J. (1998a). Nearest neighbor thermodynamic parameters for internal G.A mismatches in DNA. Biochemistry 37, 2170–2179.

    Article  PubMed  CAS  Google Scholar 

  31. Allawi, H. T. and SantaLucia, J. (1998b). Thermodynamics of internal C.T mismatches in DNA. Nucl. Acids Res. 26, 2694–2701.

    Article  PubMed  CAS  Google Scholar 

  32. Allawi, H. T. and SantaLucia, J. (1998c). Nearest neighbor thermodynamic of internal A.C mismatches in DNA. Sequence dependence and pH effects. Biochemistry 37, 9435–9444.

    Article  PubMed  CAS  Google Scholar 

  33. Peyret, N., Ananda, S. P., Allawi, H. T., and Santa Lucia, J., Jr. (1999) Nearest-neighbor thermodynamics and NMR of DNA sequences with internal A. A, C. C, G. G, and T. T, mismatches. Biochemistry 38, 3468–3477.

    Article  PubMed  CAS  Google Scholar 

  34. Krueger, J. E., Maitra, A. and Albores-Saavedra, J. (2000) Inherited medullary microcarcinoma of the thyroid: a study of 11 cases. Am. J. Surg. Pathol. 24, 853–858.

    Article  PubMed  CAS  Google Scholar 

  35. Giuffrida, D., Ferrau, F., Bordonaro, R., et al. (2000) Medullary carcinoma of the thyroid: diagnosis and therapy. Clin. Ther. 151, 29–35.

    CAS  Google Scholar 

  36. Phay, J. E., Moley, J. F. and Lairmore, T. C. (2000) Multiple endocrine neoplasias. Semin. Surg. Oncol. 18, 324–332.

    Article  PubMed  CAS  Google Scholar 

  37. Kroustrup, J. P., Laurberg, P. and Madsen, P. H. (1999) Rapid MEN2A gene carrier identification using primer-specific PCR amplification. Scand. J. Clin. Invest. 53, 642–647.

    Google Scholar 

  38. Marsh, D. J., Robinson, B. G., Andrew, S., et al. (1994) A rapid screening method for the detection of mutations in the RET proto-oncogene in multiple endocrine neoplasia type 2A and familial medullary thyroid carcinoma families. Genomics 23, 477–479.

    Article  PubMed  CAS  Google Scholar 

  39. Musholt, P. B., Musholt, T. J., Goodfellow, P. J., et al. (1997) “Cold” single-strand conformational variants for mutation analysis of the RET protooncogene. Surgery 122, 363–370.

    Article  PubMed  CAS  Google Scholar 

  40. Little, D. P., Braun, A., Darnhofer-Demor, B., et al. (1997) Detection of RET proto-oncogene codon 634 mutations using mass spectrometry. J. Mol. Med. 75, 745–750.

    Article  PubMed  CAS  Google Scholar 

  41. Kim, I.-J., Kang, H. C., Park, J.-H., et al. (2002) RET oligonucleotide microarray for the detection of RET mutations in multiple endocrine neoplasia. Clin. Cancer Res. 8, 457–463.

    PubMed  CAS  Google Scholar 

  42. Lane, M. J., Paner, T., Kashin, I., et al. (1997) The thermodynamic advantage of DNA oligonucleotide “stacking hybridization” reactions: energetics of a DNA nick. Nucleic Acids Res. 25, 611–617.

    Article  PubMed  CAS  Google Scholar 

  43. Li, Y., Zon, G., and Wilson, W. D. (1991) Thermodynamics of DNA duplexes with adjacent G·A mismatches. Biochemistry 30, 7566–7572.

    Article  PubMed  CAS  Google Scholar 

  44. Li, Y. and Agrawal, S. (1995) Oligonucleotides containing G.A pairs: effect of flanking sequences on structure and stability. Biochemistry 34, 10056–10062.

    Article  PubMed  CAS  Google Scholar 

  45. Abdoul-ela, F., Koh, D., Tinoco, I. Jr., and Martin F. H. (1985) Base-base mismatches. Thermodynamics of double helix formation for dCA3XA3G + dCT3YT3G (X, Y=A,C,G,T). Nucleic Acids Res. 13, 4811–4824.

    Article  Google Scholar 

  46. Tibanyenda, N., De Bruin, S. H., Haasnoot, C. A., et al. (1984) The effect of single base-pair mismatches on the duplex stability of d(T-A-T-T-A-A-T-A-T-C-A-A-G-T-T-G·d(C-A-A-C-T-T-G-A-T-A-T-T-A-A-T-A). Eur. J. Biochem. 139, 19–27.

    Article  PubMed  CAS  Google Scholar 

  47. Donis-Keller, H. (1995) The RET proto-oncogene and cancer. J. Intern Med. 238, 319–325.

    Article  PubMed  CAS  Google Scholar 

  48. Parinov, S., Barsky, V., Yershov, G., et al. (1996) DNA sequencing by hybridization to microchip octa- and decanucleotides extended by stacked pentanucleotides. Nucleic Acids Res. 24, 2998–3004.

    Article  PubMed  CAS  Google Scholar 

  49. Yershov, G., Barsky, V., Belgoskiy, A., et al. (1996) DNA analysis and diagnostics on oligonucleotide microchips. Proc. Natl. Acad. Sci. USA 93, 4913–4918.

    Article  PubMed  CAS  Google Scholar 

  50. Stomakhin, A.A., Vasiliskov, V.A., Timofeev, E., et al. (2000) DNA sequence analysis by hybridization with oligonucleotide microchips: MALDI mass spectrometry identification of mers contiguously stacked to microchip oligonucleotides. Nucleic Acids Res. 28, 1193–1198.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Escuela Nacional de Ciencias Biológicas, IPN, 11340, DF, México, México

    Rogelio Maldonado-Rodríguez, Mercedes Espinosa-Lara, Oscar Barrera-León, Carmen Colin-Tovar, J. Carlos Santiago-Hernández & Alfonso Méndez-Tenorio

  2. Hospital de Cancerología, CMN SXXI, IMSS, México

    Beatriz González-Yebra & Mauricio Salcedo-Vargas

  3. Molecilar Biomedicine Department, CINVESTAV-IPN, México

    Beatriz González-Yebra

  4. Life Sciences Division, Oak Ridge National Laboratory, 37831-6231, Oak Ridge, TN

    Kenneth L. Beattie

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  1. Rogelio Maldonado-Rodríguez
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  2. Mercedes Espinosa-Lara
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  4. Carmen Colin-Tovar
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  7. J. Carlos Santiago-Hernández
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  8. Alfonso Méndez-Tenorio
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  9. Kenneth L. Beattie
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Maldonado-Rodríguez, R., Espinosa-Lara, M., Barrera-León, O. et al. Detection of mutations in RET proto-oncogene codon 634 through double tandem hybridization. Mol Biotechnol 25, 113–129 (2003). https://doi.org/10.1385/MB:25:2:113

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