Skip to main content
SpringerLink
Log in

Methods for Analysis of DNA Methylation

  • Chapter
Book cover Molecular Diagnostics

Abstract

1.1. Introduction Although assessing DNA methy-lation has not yet become commonplace in the diagnostic molecular pathology laboratory, many tests involving methyla-tion analysis will become part of routine practice. In this chapter, a practical approach will be taken toward evaluating DNA methylation. The principal question likely to be asked in the pathology laboratory is whether a specific gene or region is methylated in a specific pathological situation; for example, is the MLH1 gene promoter methylated in a colorectal cancer specimen showing microsatellite instability? This chapter will deal with methods that examine specific sequences for methy-lation rather than those examining total genomic methylation or those screening for new sites of recurrent methylation.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 189.00
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Gardiner-Garden, M. and Frommer, M. CpG islands in vertebrate genomes. J. Mol. Biol. 196:261–282, 1987.

    Article  Google Scholar 

  2. Cooper, D. N. and Krawczak, M. Cytosine methylation and the fate of CpG dinucleotides in vertebrate genomes. Hum. Genet. 83: 181–188, 1989.

    Article  PubMed  CAS  Google Scholar 

  3. Krawczak, M., Ball, E. V., and Cooper, D. N. Neighboring-nucleotide effects on the rates of germ-line single-base-pair substitution in human genes. Am. J. Hum. Genet. 63:474–488, 1998.

    Article  PubMed  CAS  Google Scholar 

  4. Bird, A. P. CpG-rich islands and the function of DNA methylation. Nature 321:209–213, 1986.

    Article  PubMed  CAS  Google Scholar 

  5. Nan, X., Ng, H. H., Johnson, C. A., et al. Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393:386–389, 1998.

    Article  PubMed  CAS  Google Scholar 

  6. Jones, P. L., Veenstra, G. J., Wade, P. A., et al. Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat. Genet. 19:187–191, 1998.

    Article  PubMed  CAS  Google Scholar 

  7. Ballestar, E. and Wolffe, A. P. Methyl-CpG-binding proteins. Targeting specific gene repression. Eur. J. Biochem. 268:1–6, 2001.

    Article  PubMed  CAS  Google Scholar 

  8. Hendrich, B., Hardeland, U., Ng, H. H., Jiricny, J., and Bird, A. The thymine glycosylase MBD4 can bind to the product of deamination at methylated CpG sites. Nature 401:301–304, 1991.

    Google Scholar 

  9. Li, E., Bestor, T. H., and Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69:915–926, 1992.

    Article  PubMed  CAS  Google Scholar 

  10. Okano, M., Bell, D. W., Haber, D. A., and Li, E. DNA methyltrans-ferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99:247–257, 1991.

    Article  Google Scholar 

  11. Dong, A., Yoder, J. A., Zhang, X., Zhou, L., Bestor, T. H., and Cheng, X. Structure of human DNMT2, an enigmatic DNA methyl-transferase homolog that displays denaturant-resistant binding to DNA. Nucleic Acids Res. 29:439–448, 2001.

    Article  PubMed  CAS  Google Scholar 

  12. Hata, K., Okano, M., Lei, H., and Li, E. Dnmt3L cooperates with the Dnmt3 family of de novo DNA methyltransferases to establish maternal imprints in mice. Development 129:1983–1993, 2002.

    PubMed  CAS  Google Scholar 

  13. Chedin, F., Lieber, M. R., and Hsieh, C. L. The DNA methyltrans-ferase-like protein DNMT3L stimulates de novo methylation by Dnmt3a. Proc. Natl. Acad. Sci. USA 99:16,916–16,921, 2002.

    Article  PubMed  CAS  Google Scholar 

  14. Holliday, R. and Pugh, J. E. DNA modification mechanisms and gene activity during development. Science 187:226–232, 1975.

    Article  PubMed  CAS  Google Scholar 

  15. Riggs, A. D. X inactivation, differentiation and DNA methylation. Cytogenet. Cell. Genet. 14:9–25, 1975.

    Article  PubMed  CAS  Google Scholar 

  16. Razin, A. and Shemer, R. DNA methylation in early development. Hum. Mol. Genet. 4:1751–1755, 1995.

    PubMed  CAS  Google Scholar 

  17. Behn-Krappa, A., Holker, I., Sandaradura de Silva, U., and Doerfler, W. Patterns of DNA methylation are indistinguishable in different individuals over a wide range of human DNA sequences. Genomics 11:1–7, 1991.

    Article  PubMed  CAS  Google Scholar 

  18. Robertson, K. D. and Wolffe, A. P. DNA methylation in health and disease. Nat. Rev. Genet. 1: 11–19, 2000.

    Article  PubMed  CAS  Google Scholar 

  19. Xu, G. L., Bestor, T. H., Bourc’his, D., et al. Chromosome instability and immunodeficiency syndrome caused by mutations in a DNA methyltransferase gene. Nature 402:187–191, 1999.

    Article  PubMed  CAS  Google Scholar 

  20. Hansen, R. S., Wijmenga, C., Luo, P., The DNMT3B DNA methyl-transferase gene is mutated in the ICF immunodeficiency syndrome. Proc. Natl. Acad. Sci. USA 96:14,412–14,417, 1999.

    Article  PubMed  CAS  Google Scholar 

  21. Fryns, J. P., Azou, M., Jaeken, J., Eggermont, E., Pedersen, J. C., and Van den Berghe, H. Centromeric instability of chromosomes 1, 9 and 16 associated with combined immunodeficiency. Hum. Genet. 57:108–110, 1981.

    PubMed  CAS  Google Scholar 

  22. Haas, O. Centromeric heterochromatin instability of chromosomes 1, 9 and 16 in variable immunodeficiency syndrome––a virus-induced phenomenon? Hum. Genet. 85:244–246, 1990.

    Article  PubMed  CAS  Google Scholar 

  23. Ehrlich, M. DNA hypomethylation, cancer, the immunodeficiency, centromeric region instability, facial anomalies syndrome and chromosomal rearrangements. J. Nutr. 132:2424S–2429S, 2002.

    PubMed  CAS  Google Scholar 

  24. Gibbons, R. J. and Higgs, D. R. Molecular-clinical spectrum of the ATR-X syndrome. Am. J. Med. Genet. 97:204–212, 2002.

    Article  Google Scholar 

  25. Gibbons, R. J., McDowell, T. L., Raman, S., et al. Mutations in ATRX, encoding a SWI/SNF-like protein, cause diverse changes in the pattern of DNA methylation. Nat. Genet. 24:368–371, 2000.

    Article  PubMed  CAS  Google Scholar 

  26. Shahbazian, M. D. and Zoghbi, H. Y. Rett syndrome and MeCP2: linking epigenetics and neuronal function. Am. J. Hum. Genet. 71:1259–1272, 2002.

    Article  PubMed  CAS  Google Scholar 

  27. Oberle, I., Rousseau, F., Heitz, D., et al. Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome. Science 2521097–1102, 1991.

    Article  CAS  Google Scholar 

  28. Kremer, E. J., Pritchard, M., Lynch, M., et al. Mapping of DNA instability at the fragile X to a trinucleotide repeat sequence p(CCG)n. Science 252:1711–1714, 1991.

    Article  PubMed  CAS  Google Scholar 

  29. Gecz, J. The FMR2 gene, FRAXE and non-specific X-linked mental retardation: clinical and molecular aspects. Ann. Hum. Genet. 64:95–106, 2000.

    Article  PubMed  CAS  Google Scholar 

  30. Gecz, J. FMR3 is a novel gene associated with FRAXE CpG island and transcriptionally silent in FRAXE full mutations. J. Med. Genet. 37:782–784, 2000.

    Article  PubMed  CAS  Google Scholar 

  31. Jones, P. A. and Baylin, S. B. The fundamental role of epigenetic events in cancer. Nat. Rev. Genet. 3:15–428, 2002.

    Article  CAS  Google Scholar 

  32. Costello, J. F., Fruhwald, M. C., Smiraglia, D. J., et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat. Genet. 24:132–138, 2000.

    Article  PubMed  CAS  Google Scholar 

  33. Greger, V., Passarge, E., Hopping, W., Messmer, E., and Horsthemke, B. Epigenetic changes may contribute to the formation and spontaneous regression of retinoblastoma. Hum. Genet. 83:155–158, 1989.

    Article  PubMed  CAS  Google Scholar 

  34. Sakai, T., Toguchida, J., Ohtani, N., Yandell, D. W., Rapaport, J. M., and Dryja, T. P. Allele-specific hypermethylation of the retinoblas-toma tumor-suppressor gene. Am. J. Hum. Genet. 48:880–888, 1991.

    PubMed  CAS  Google Scholar 

  35. Stirzaker, C., Millar, D. S., Paul, C. L., et al. Extensive DNA methy-lation spanning the Rb promoter in retinoblastoma tumors. Cancer Res. 57:2229–2237, 1997.

    PubMed  CAS  Google Scholar 

  36. Herman, J. G., Latif, F., Weng, Y., et al. Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc. Natl. Acad. Sci. USA. 91:9700–9704, 1994.

    Article  PubMed  CAS  Google Scholar 

  37. Kane, M. F., Loda, M., Gaida, G. M., et al. Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res. 57:808–811, 1997.

    PubMed  CAS  Google Scholar 

  38. Herman, J. G., Umar, A., Polyak, K., et al. Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc. Natl. Acad. Sci. USA 95:6870–6875, 1998.

    Article  PubMed  CAS  Google Scholar 

  39. Bianco, T., Chenevix-Trench, G., Walsh, D. C., Cooper, J. E., and Dobrovic, A. Tumour-specific distribution of BRCA1 promoter region methylation supports a pathogenetic role in breast and ovarian cancer. Carcinogenesis 21:147–151, 2000.

    Article  PubMed  CAS  Google Scholar 

  40. Esteller, M., Silva, J. M., Dominguez, G., et al. Promoter hyperme-thylation and BRCA1 inactivation in sporadic breast and ovarian tumors. J. Natl. Cancer Inst. 92:564–569, 2000.

    Article  PubMed  CAS  Google Scholar 

  41. Hedenfalk, I., Duggan, D., Chen, Y., et al. Gene-expression profiles in hereditary breast cancer. N. Engl. J. Med. 344:539–548, 2001.

    Article  PubMed  CAS  Google Scholar 

  42. Collins, N., Wooster, R., and Stratton, M. R. Absence of methyla-tion of CpG dinucleotides within the promoter of the breast cancer susceptibility gene BRCA2 in normal tissues and in breast and ovarian cancers. Br. J. Cancer 76:1150–1156, 1997.

    PubMed  CAS  Google Scholar 

  43. Yan, P. S., Chen, C. M., Shi, H., et al. Dissecting complex epigenetic alterations in breast cancer using CpG island microarrays. Cancer Res. 61:8375–8380, 2001.

    PubMed  CAS  Google Scholar 

  44. Toyota, M., Ahuja, N., Ohe-Toyota, M., et al. CpG island methyla-tor phenotype in colorectal cancer. Proc. Natl. Acad. Sci. USA 96:8681–8686, 2001.

    Article  Google Scholar 

  45. Santini, V., Kantarjian, H. M., and Issa, J. P. Changes in DNA methylation in neoplasia: pathophysiology and therapeutic implications. Ann. Intern. Med. 134:573–586, 2001.

    PubMed  CAS  Google Scholar 

  46. List, A. F. New approaches to the treatment of myelodysplasia. Oncologist 7( Suppl. 1):39–49, 2002.

    Article  PubMed  Google Scholar 

  47. Daskalakis, M., Nguyen, T. T., Nguyen, C., et al. Demethylation of a hypermethylated P15/INK4B gene in patients with myelodysplas-tic syndrome by 5-Aza-2′-deoxycytidine (decitabine) treatment. Blood 100:2957–2964, 2002.

    Article  PubMed  CAS  Google Scholar 

  48. Leone, G., Teofili, L., Voso, M. T., and Lubbert, M. DNA methyla-tion and demethylating drugs in myelodysplastic syndromes and secondary leukemias. Haematologica 87:1324–1341, 2002.

    PubMed  CAS  Google Scholar 

  49. Wang, R. Y., Gehrke, C. W., and Ehrlich, M. Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues. Nucleic Acids Res. 8:4777–4790, 1980.

    Article  PubMed  CAS  Google Scholar 

  50. Frommer, M., Mcdonald, L. E., Millar, D. S., et al. A Genomic Sequencing Protocol That Yields a Positive Display of 5-Methylcytosine Residues in Individual DNA Strands. Proc. Natl. Acad.Sci. USA 89:1827–1831, 1992.

    Article  PubMed  CAS  Google Scholar 

  51. Clark, S. J., Harrison, J., Paul, C. L., and Frommer, M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22:2990–2997, 1994.

    Article  PubMed  CAS  Google Scholar 

  52. Clark, S. J. and Frommer, M. F. Deamination with NaHSO3 in DNA methylation studies, in DNA and Nucleoprotein Structure InVivo, Saluz, H. and Wiebauer, K., eds., Biomedical Publishers. pp. 123–132, 1995.

    Google Scholar 

  53. Grunau, C., Clark, S. J., and Rosenthal, A. Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res. 29:E65,5, 2001.

    Article  PubMed  CAS  Google Scholar 

  54. Warnecke, P. M., Stirzaker, C., Song, J., Grunau, C., Melki, J. R., and Clark, S. J. Identification and resolution of artifacts in bisulfite sequencing. Methods. 27:101–107, 2002.

    Article  PubMed  CAS  Google Scholar 

  55. McDonald, L. E. and Kay, G. F. Methylation analysis using bisulfite genomic sequencing: application to small numbers of intact cells. Biotechniques 22:272–274, 1997.

    PubMed  CAS  Google Scholar 

  56. Bian, Y. S., Yan, P., Osterheld, M. C., Fontolliet, C., and Benhattar, J. Promoter methylation analysis on microdissected paraffin-embedded tissues using bisulfite treatment and PCR-SSCP. Biotechniques 30:66–72, 2001.

    PubMed  CAS  Google Scholar 

  57. Tan, L. W. and Dobrovic, A. Methylation analysis of formalin-fixed, paraffin-embedded sections using a nontoxic DNA extraction protocol. Biotechniques 31:1354, 1356–1357, 2001.

    Google Scholar 

  58. Millar, D. S., Warnecke, P. M., Melki, J. R., and Clark, S. J. Methylation sequencing from limiting DNA: embryonic, fixed and microdissected cells. Methods 27:108–113, 2002.

    Article  PubMed  CAS  Google Scholar 

  59. Paul, C. L. and Clark, S. J. Cytosine methylation: quantitation by automated genomic sequencing and GENESCAN analysis. Biotechniques 21:126–133, 1996.

    PubMed  CAS  Google Scholar 

  60. Herman, J. G., Graff, J. R., Myohanen, S., Nelkin, B. D., and Baylin, S. B. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. USA 93:9821–9826, 1996.

    Article  PubMed  CAS  Google Scholar 

  61. Wu, D. Y., Ugozzoli, L., Pal, B. K., and Wallace, R. B. Allele-specific enzymatic amplification of beta-globin genomic DNA for diagnosis of sickle cell anemia. Proc. Natl. Acad. Sci. USA 86:2757–2760, 1989.

    Article  PubMed  CAS  Google Scholar 

  62. Newton, C. R., Graham, A., Heptinstall, L. E., et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 17:2503–2516, 1989.

    Article  PubMed  CAS  Google Scholar 

  63. Sarkar, G., Cassady, J., Bottema, C., and Sommer, S. S. Characterization of polymerase chain reaction amplification of specific alleles. Anal. Biochem. 186:64–68, 1998.

    Article  Google Scholar 

  64. Dodge, J. E., List, A. F., and Futscher, B. W. Selective variegated methylation of the p15 CpG island in acute myeloid leukemia. Int. J. Cancer 78:561–567, 1998.

    Article  PubMed  CAS  Google Scholar 

  65. Tan, L. W., Bianco, T., and Dobrovic, A. Variable promoter region CpG island methylation of the putative tumor suppressor gene Connexin 26 in breast cancer. Carcinogenesis 23:231–236, 2002.

    Article  PubMed  CAS  Google Scholar 

  66. Aggerholm, A. and Hokland, P. DAP-kinase CpG island methyla-tion in acute myeloid leukemia: methodology versus biology? Blood 95:2997–2999, 2000.

    PubMed  CAS  Google Scholar 

  67. Aggerholm, A., Guldberg, P., Hokland, M., and Hokland, P. Extensive intra- and interindividual heterogeneity of p15INK4B methylation in acute myeloid leukemia. Cancer Res. 59:436–441, 1999.

    PubMed  CAS  Google Scholar 

  68. Xiong, Z. and Laird, P. W. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 25:2532–2534, 1997.

    Article  PubMed  CAS  Google Scholar 

  69. Eads, C. A. and Laird, P. W. Combined bisulfite restriction analysis (COBRA). Methods Mol. Biol. 200:71–85, 2002.

    PubMed  CAS  Google Scholar 

  70. Sadri, R. and Hornsby, P. J. Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification. Nucleic Acids Res. 24:5058–5059, 1996.

    Article  PubMed  CAS  Google Scholar 

  71. Uejima, H., Lee, M. P., Cui, H., and Feinberg, A. P. Hot-stop PCR: a simple and general assay for linear quantitation of allele ratios. Nat. Genet. 25:375–376, 2000.

    Article  PubMed  CAS  Google Scholar 

  72. Kuppuswamy, M. N., Hoffmann, J. W., Kasper, C. K., Spitzer, S. G., Groce, S. L., and Bajaj, S. P. Single nucleotide primer extension to detect genetic diseases: experimental application to hemophilia B (factor IX) and cystic fibrosis genes. Proc. Natl. Acad. Sci. USA 88:1143–1147, 1991.

    Article  PubMed  CAS  Google Scholar 

  73. Gonzalgo, M. L. and Jones, P. A. Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 25:2529–2531, 1997.

    Article  PubMed  CAS  Google Scholar 

  74. Gonzalgo, M. L. and Jones, P. A. Quantitative methylation analysis using methylation-sensitive single-nucleotide primer extension (Ms-SNuPE). Methods 27:128–133, 2002.

    Article  PubMed  CAS  Google Scholar 

  75. El-Maarri, O., Herbiniaux, U., Walter, J., and Oldenburg, J. A rapid, quantitative, non-radioactive bisulfite-SNuPE- IP RP HPLC assay for methy-lation analysis at specific CpG sites. Nucleic Acids Res. 30:e25, 2002.

    Article  PubMed  Google Scholar 

  76. Galm, O., Rountree, M. R., Bachman, K. E., Jair, K. W., Baylin, S. B., and Herman, J. G. Enzymatic regional methylation assay: a novel method to quantify regional CpG methylation density. Genome Res. 12:153–157, 2002.

    Article  PubMed  CAS  Google Scholar 

  77. Cameron, E. E., Baylin, S. B., and Herman, J. G. p15(INK4B) CpG island methylation in primary acute leukemia is heterogeneous and suggests density as a critical factor for transcriptional silencing. Blood 94:2445–2451, 1999.

    PubMed  CAS  Google Scholar 

  78. Burri, N. and Chaubert, P. Complex methylation patterns analyzed by single-strand conformation polymorphism. Biotechniques 26:232–234, 1999.

    PubMed  CAS  Google Scholar 

  79. Bianco, T., Hussey, D., and Dobrovic, A. Methylation-sensitive, single-strand conformation analysis (MS-SSCA): A rapid method to screen for and analyze methylation. Hum. Mutat. 14:289–293, 1999.

    Article  PubMed  CAS  Google Scholar 

  80. Maekawa, M., Sugano, K., Ushiama, M., et al. Heterogeneity of DNA methylation status analyzed by bisulfite–PCR–SSCP and correlation with clinico-pathological characteristics in colorectal cancer. Clin. Chem. Lab. Med. 39:121–128, 2001.

    Article  PubMed  CAS  Google Scholar 

  81. Dobrovic, A., Bianco, T., Tan, L. W., Sanders, T., and Hussey, D. Screening for and analysis of methylation differences using methy-lation-sensitive single-strand conformation analysis. Methods 27:134–138, 2002.

    Article  PubMed  CAS  Google Scholar 

  82. Liu, Q., Feng, J., Buzin, C., et al. Detection of virtually all muta-tions-SSCP (DOVAM-S): a rapid method for mutation scanning with virtually 100% sensitivity. Biotechniques 26:932, 936–938, 940–942, 1999.

    PubMed  CAS  Google Scholar 

  83. Guldberg, P., Worm, J., and Gronbaek, K. Profiling DNA methyla-tion by melting analysis. Methods 27:121–127,2002.

    Article  PubMed  CAS  Google Scholar 

  84. Worm, J., Aggerholm, A., and Guldberg, P. In-tube DNA methyla-tion profiling by fluorescence melting curve analysis. Clin. Chem. 47:1183–1189, 2001.

    PubMed  CAS  Google Scholar 

  85. Baumer, A., Wiedemann, U., Hergersberg, M., and Schinzel, A. A novel MSP/DHPLC method for the investigation of the methylation status of imprinted genes enables the molecular detection of low cell mosaicisms. Hum. Mutat. 17:423–430, 2001.

    Article  PubMed  CAS  Google Scholar 

  86. Deng, D. J., Zhou, J., Zhu, B. D., Ji, J. F., Harper, J. C., and Powell, S. M. Silencing-specific methylation and single nucleotide polymorphism of hMLH1 promoter in gastric carcinomas. World J. Gastroenterol. 9:26–29, 2003.

    PubMed  CAS  Google Scholar 

  87. Eads, C. A., Danenberg, K. D., Kawakami, K., et al. MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res. 28:E32, 2000.

    Article  PubMed  CAS  Google Scholar 

  88. Trinh, B. N., Long, T. I., and Laird, P.,W. DNA methylation analysis by MethyLight technology. Methods 25:456–462, 2001.

    Article  PubMed  CAS  Google Scholar 

  89. Rand, K., Qu, W., Ho, T., Clark, S. J., and Molloy, P. Conversion-specific detection of DNA methylation using real-time polymerase chain reaction (ConLight-MSP) to avoid false positives. Methods 27:114–120, 2002.

    Article  PubMed  CAS  Google Scholar 

  90. Rush, L. J. and Plass, C. Restriction landmark genomic scanning for DNA methylation in cancer: past, present and future applications. Anal. Biochem. 307:191–201, 2002.

    Article  PubMed  CAS  Google Scholar 

  91. Costello, J. F., Plass, C., and Cavenee, W. K. Restriction landmark genome scanning. Methods Mol. Biol. 200:53–70, (2002).

    PubMed  CAS  Google Scholar 

  92. Huang, T. H., Perry, M. R. and Laux, D. E. Methylation profiling of CpG islands in human breast cancer cells. Hum. Mol. Genet. 8:459–470, 1999.

    Article  PubMed  CAS  Google Scholar 

  93. Balog, R. P., Emi Ponce de Souza, Y., Tang, H. M., et al. Parallel assessment of CpG methylation by two-color hybridization with oligonucleotide arrays. Anal. Biochem. 309:301–310, 2002.

    Article  PubMed  CAS  Google Scholar 

  94. Shi, H., Maier, S., Nimmrich, I., et al. Oligonucleotide-based microarray for DNA methylation analysis: principles and applications. J. Cell Biochem. 88:138–143, 2003.

    Article  PubMed  CAS  Google Scholar 

  95. Li, L. C. and Dahiya, R. MethPrimer: designing primers for methy-lation PCRs. Bioinformatics 18:1427–1431, 2002.

    Article  PubMed  CAS  Google Scholar 

  96. Singal, R. and Grimes, S. R. Microsoft Word macro for analysis of cytosine methylation by the bisulfite deamination reaction. Biotechniques 30:116–120, 2001.

    PubMed  CAS  Google Scholar 

  97. Esteller, M., Corn, P. G., Baylin, S. B., and Herman, J. G. A gene hypermethylation profile of human cancer. Cancer Res. 61:3225–3229, 2001.

    PubMed  CAS  Google Scholar 

  98. Laird, P. W. Early detection: the power and the promise of DNA methylation markers. Nat. Rev. Cancer. 3:253–266, 2003.

    Article  PubMed  CAS  Google Scholar 

  99. Lee, W. H., Isaacs, W. B., Bova, G. S., and Nelson, W. G. CG island methylation changes near the GSTP1 gene in prostatic carcinoma cells detected using the polymerase chain reaction: a new prostate cancer biomarker. Cancer Epidemiol. Biomarkers Prev. 6:443–450, 1997.

    PubMed  CAS  Google Scholar 

  100. Wong, I. H., Lo, Y. M., and Johnson, P. J. Epigenetic tumor markers in plasma and serum: biology and applications to molecular diagnosis and disease monitoring. Ann. N Y Acad. Sci. 945:36–50, 2001.

    Article  PubMed  CAS  Google Scholar 

  101. Jahr, S., Hentze, H., Englisch, S.,et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 61:1659–1665, 2001.

    PubMed  CAS  Google Scholar 

  102. Belinsky, S. A., Nikula, K. J., Palmisano, W. A., et al. Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis. Proc. Natl. Acad. Sci. USA 95:11,891–11,896, 1998.

    Article  PubMed  CAS  Google Scholar 

  103. Ahrendt, S. A., Chow, J. T., Xu, L. H., et al. Molecular detection of tumor cells in bronchoalveolar lavage fluid from patients with early stage lung cancer. J. Natl. Cancer Inst. 91:332–339, 1999.

    Article  PubMed  CAS  Google Scholar 

  104. Goessl, C., Muller, M., Straub, B., and Miller, K. DNA alterations in body fluids as molecular tumor markers for urological malignancies. Eur. Urol. 41:668–676, 2002.

    Article  PubMed  CAS  Google Scholar 

  105. Evron, E., Dooley, W. C., Umbricht, C. B., et al. Detection of breast cancer cells in ductal lavage fluid by methylation-specific PCR. Lancet 357:1335–1336, 2001.

    Article  PubMed  CAS  Google Scholar 

  106. Vogelstein, B., Fearon, E. R., Hamilton, S. R., et al. Clonal analysis using recombinant DNA probes from the X-chromosome. Cancer Res. 47:4806–48013, 1987.

    PubMed  CAS  Google Scholar 

  107. van Kamp, H., Jansen, R., Willemze, et al. Studies on clonality by PCR analysis of t he PGK-1 gene. Nucleic Acids Res. 19:2794, 1991.

    Article  Google Scholar 

  108. Uchida, T., Ohashi, H., Aoki, E., et al. Clonality analysis by methy-lation-specific PCR for the human androgen-receptor gene (HUMARA-MSP). Leukemia 14:207–212, 2000.

    Article  PubMed  CAS  Google Scholar 

  109. Nakahara, Y., Suzuki, H., Ohashi, H., et al. Clonality analysis of granulocytes and T lymphocytes in healthy females by the PCR-based HUMARA method. Int. J. Hematol. 69:237–243.

    Google Scholar 

  110. Kubota, T., Das, S., Christian, S. L., Baylin, S. B., Herman, J. G. and Ledbetter, D. H. (1997) Methylation-specific PCR simplifies imprinting analysis. Nat. Genet. 16:16–17, 1999.

    Google Scholar 

  111. Zeschnigk, M., Lich, C., Buiting, K., Doerfler, W., and Horsthemke, B. A single-tube PCR test for the diagnosis of Angelman and Prader–Willi syndrome based on allelic methylation differences at the SNRPN locus. Eur. J. Hum. Genet. 5:94–98, 1997.

    PubMed  CAS  Google Scholar 

  112. Kosaki, K., McGinniss, M. J., Veraksa, A. N., McGinnis, W. J., and Jones, K. L. Prader–Willi and Angelman syndromes: diagnosis with a bisulfite-treated methylation-specific PCR method. Am. J. Med. Genet. 73:308–313, 1997.

    Article  PubMed  CAS  Google Scholar 

  113. Baumer, A. Analysis of the methylation status of imprinted genes based on methylation-specific polymerase chain reaction combined with denaturing high-performance liquid chromatography. Methods 27:139–143, 2002.

    Article  PubMed  CAS  Google Scholar 

  114. Couvert, P., Poirier, K., Carrie, A., DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes. Biotechniques 34:356–362, 2003.

    PubMed  CAS  Google Scholar 

  115. Esteller, M., Hamilton, S. R., Burger, P. C., Baylin, S. B., and Herman, J. G. Inactivation of the DNA repair gene O6-methylgua-nine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res. 59:793–797, 1999.

    PubMed  CAS  Google Scholar 

  116. Esteller, M., Garcia-Foncillas, J., Andion, E.,et al. Inactivation of the DNA-repair gene MGMT and the clinical response of gliomas to alkylating agents. N. Engl. J. Med. 343:1350–1354, 2000.

    Article  PubMed  CAS  Google Scholar 

  117. Esteller, M., Gaidano, G., Goodman, S. N., et al. Hypermethylation of the DNA repair gene O(6)-methylguanine DNA methyltrans-ferase and survival of patients with diffuse large B-cell lymphoma. J. Natl. Cancer Inst. 94:26–32, 2002.

    PubMed  CAS  Google Scholar 

  118. Li, G. M. The role of mismatch repair in DNA damage-induced apoptosis. Oncol. Res. 11:393–400, 1999.

    PubMed  CAS  Google Scholar 

  119. Elsaleh, H., Shannon, B., and Iacopetta, B. Microsatellite instability as a molecular marker for very good survival in colorectal cancer patients receiving adjuvant chemotherapy. Gastroenterology 120:1309–1310, 2001.

    Article  PubMed  CAS  Google Scholar 

  120. Hemminki, A., Mecklin, J. P., Jarvinen, H., Aaltonen, L. A., and Joensuu, H. Microsatellite instability is a favorable prognostic indicator in patients with colorectal cancer receiving chemotherapy. Gastroenterology 119:921–928, 2000.

    Article  PubMed  CAS  Google Scholar 

  121. Whitehall, V. L., Walsh, M. D., Young, J., Leggett, B. A., and Jass, J. R. Methylation of O-6-methylguanine DNA methyltransferase characterizes a subset of colorectal cancer with low-level DNA microsatellite instability. Cancer Res. 61:827–830, 2001.

    PubMed  CAS  Google Scholar 

  122. Jass, J. R., Walsh, M. D., Barker, M., Simms, L. A., Young, J., and Leggett, B.A. Distinction between familial and sporadic forms of colorectal cancer showing DNA microsatellite instability. Eur. J. Cancer 38:858–866, 2002.

    Article  PubMed  CAS  Google Scholar 

  123. Boland, C. R., Thibodeau, S. N., Hamilton, S. R., et al. A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorec-tal cancer. Cancer Res. 58:5248–5257, 1998.

    PubMed  CAS  Google Scholar 

  124. Warnecke, P. M., Stirzaker, C., Melki, J. R., Millar, D. S., Paul, C. L., and Clark, S. J. Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA. Nucleic Acids Res. 25: 4422–4426, 1997.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Australia

    Alexander Dobrovic PhD

Authors
  1. Alexander Dobrovic PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Pathology and Laboratory Medicine, University of North Carolina School of Medicine, Chapel Hill, NC, USA

    William B. Coleman

  2. Department of Pathology, Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA

    Gregory J. Tsongalis

Rights and permissions

Reprints and permissions

Copyright information

© 2006 Humana Press, a part of Springer Science+Business Media, LLC

About this chapter

Cite this chapter

Dobrovic, A. (2006). Methods for Analysis of DNA Methylation. In: Coleman, W.B., Tsongalis, G.J. (eds) Molecular Diagnostics. Humana Press. https://doi.org/10.1385/1-59259-928-1:149

Download citation

  • DOI: https://doi.org/10.1385/1-59259-928-1:149

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-356-5

  • Online ISBN: 978-1-59259-928-8

  • eBook Packages: MedicineMedicine (R0)

Publish with us

Policies and ethics

Search

Navigation