Bisulfite sequencing

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Bisulfite conversion

The bisulfite sequencing is a biochemical method for determination of DNA methylation in the context of epigenetic investigations by reaction with bisulfite . Bisulfite sequencing consists of bisulfite conversion, mostly in combination with DNA sequencing .

principle

DNA methylation occurs in epigenetics to inactivate genes . In animals, methylation primarily affects cytosines at the C5 position in CpG dinucleotides . By treating DNA with bisulfite, the bisulfite reaction converts unmethylated cytosines to uracils , while 5-methylcytosine does not react with bisulfite. A subsequent amplification usually takes place via polymerase chain reaction (PCR), via methylation-specific PCR (MSP) or via microarray . Problems with bisulfite sequencing are the inability to differentiate between methylcytosine and the hydroxymethylcytosine that occurs in some tissues, as well as an incomplete bisulfite reaction in double-stranded DNA and increased breakdown of DNA by depurination . Uracil is recognized in the PCR and DNA sequencing like thymine and the opposite strand is filled with adenine due to the base pairing , while in the bisulfite-untreated DNA sequencing cytidines are paired with guanines. This results in two DNA sequences (treated and untreated), of which the bisulfite-treated person has C-> U transitions at the unmethylated sites and which is then paired with A instead of G in a DNA sequencing. Cytosines can be clearly determined by previous oxidation of hydroxymethylcytosines.

PCR-based analysis methods

PCR-based analysis methods are e.g. B. DNA sequencing , methylation-sensitive single-strand conformation analysis (MS-SSCA), high resolution melting analysis (HRM), methylation-sensitive single-nucleotide primer extension (MS-SnuPE) and base-specific cleavage / MALDI-TOF .

Methylation-specific PCR variants are e.g. B. MSP, methylation- sensitive qPCR MethyLight , melting curve analysis (Mc-MSP) and oligonucleotide microarrays.

Alternative methods are e.g. B. the Combined Bisulphite Restriction Analysis and the methylated DNA immunoprecipitation (MeDIP).

Web links

Individual evidence

  1. ↑ Frommer M, McDonald LE, Millar DS, et al. : A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands . In: Proc. Natl. Acad. Sci. USA . 89, No. 5, March 1992, pp. 1827-31. doi : 10.1073 / pnas.89.5.1827 . PMID 1542678 . PMC 48546 (free full text).
  2. ↑ Chatterjee A, Stockwell PA, Rodger EJ and Morison IM 2012. Comparison of alignment software for genome-wide bisulphite sequence data. Nucleic Acids Research 40 (10): e79.
  3. ↑ Fraga MF, Esteller M: DNA methylation: a profile of methods and applications . In: BioTechniques . 33, No. 3, September 2002, pp. 632, 634, 636-49. PMID 12238773 .
  4. ↑ Osman El-Maarri: Methods: DNA methylation . In: Peroxisomal Disorders and Regulation of Genes (=  Advances in Experimental Medicine and Biology ). tape 544 , 2003, ISBN 978-0-306-48174-1 , pp. 197-204 , doi : 10.1007 / 978-1-4419-9072-3_23 .
  5. ↑ Laird PW: The power and the promise of DNA methylation markers . In: Nat. Rev. Cancer . 3, No. 4, April 2003, pp. 253-66. doi : 10.1038 / nrc1045 . PMID 12671664 .
  6. ↑ Callinan PA, Feinberg AP: The emerging science of epigenomics . In: Hum Mol Genet . 15, No. 90001, 2006, pp. R95-101. doi : 10.1093 / hmg / ddl095 .
  7. ↑ Huang Y, Pastor WA, Shen Y, Tahiliani M, Liu DR, Rao A. The Behavior of 5-Hydroxymethylcytosine in Bisulfite Sequencing. PloS one.2010; 5 (1): e8888 .
  8. ↑ Grunau C, Clark SJ, Rosenthal A: Bisulfite genomic sequencing: systematic investigation of critical experimental parameters . In: Nucleic Acids Res . 29, No. 13, July 2001, pp. E65-5. doi : 10.1093 / nar / 29.13.e65 . PMID 11433041 . PMC 55789 (free full text).
  9. ↑ Ehrich M, Zoll S, Sur S, van den Boom D: A new method for accurate assessment of DNA quality after bisulfite treatment . In: Nucleic Acids Res . 35, No. 5, 2007, p. 29. doi : 10.1093 / nar / gkl1134 . PMID 17259213 . PMC 1865059 (free full text).
  10. ↑ MJ Booth, MR Branco, G. Ficz, D. Oxley, F. Krueger, W. Reik, S. Balasubramanian: Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. In: Science. Volume 336, Number 6083, May 2012, pp. 934-937, ISSN  1095-9203 . doi : 10.1126 / science.1220671 . PMID 22539555 .
  11. ↑ Colella S, Shen L, Baggerly KA, Issa JP, Krahe R: Sensitive and quantitative universal Pyrosequencing methylation analysis of CpG sites . In: BioTechniques . 35, No. 1, July 2003, pp. 146-50. PMID 12866414 .
  12. ↑ Tost J, Dunker J, Gut IG: Analysis and quantification of multiple methylation variable positions in CpG islands by Pyrosequencing . In: BioTechniques . 35, No. 1, July 2003, pp. 152-6. PMID 12866415 .
  13. ↑ Bianco T, Hussey D, Dobrovic A: Methylation-sensitive, single-strand conformation analysis (MS-SSCA): A rapid method to screen for and analyze methylation . In: Hum. Mutat. . 14, No. 4, 1999, pp. 289-93. doi : 10.1002 / (SICI) 1098-1004 (199910) 14: 4 <289 :: AID-HUMU3> 3.0.CO; 2-A . PMID 10502775 .
  14. ↑ Wojdacz TK, Dobrovic A: Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation . In: Nucleic Acids Res . 35, No. 6, 2007, p. E41. doi : 10.1093 / nar / gkm013 . PMID 17289753 . PMC 1874596 (free full text).
  15. ↑ Gonzalgo ML, Jones PA: Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE) . In: Nucleic Acids Res . 25, No. 12, June 1997, pp. 2529-31. doi : 10.1093 / nar / 25.12.2529 . PMID 9171109 . PMC 146734 (free full text).
  16. ↑ Uhlmann K, Brinckmann A, Toliat MR, Ritter H, Nürnberg P: Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis . In: Electrophoresis . 23, No. 24, December 2002, pp. 4072-9. doi : 10.1002 / elps.200290023 . PMID 12481262 .
  17. ↑ Ehrich M, Nelson MR, Stanssens P, et al. : Quantitative high-throughput analysis of DNA methylation patterns by base-specific cleavage and mass spectrometry . In: Proc. Natl. Acad. Sci. USA . 102, No. 44, November 2005, pp. 15785-90. doi : 10.1073 / pnas.0507816102 . PMID 16243968 . PMC 1276092 (free full text).
  18. ↑ Frommer M, McDonald LE, Millar DS, et al. (March 1992). "A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands". Proc. Natl. Acad. Sci. USA 89 (5): 1827-31. doi : 10.1073 / pnas.89.5.1827 . PMC 48546 (free full text). PMID 1542678 .
  19. ↑ Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB: Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands . In: Proc. Natl. Acad. Sci. USA . 93, No. 18, September 1996, pp. 9821-6. doi : 10.1073 / pnas.93.18.9821 . PMID 8790415 . PMC 38513 (free full text).
  20. ↑ Eads CA, Danenberg KD, Kawakami K, et al. : MethyLight: a high-throughput assay to measure DNA methylation . In: Nucleic Acids Res . 28, No. 8, April 2000, p. E32. doi : 10.1093 / nar / 28.8.e32 . PMID 10734209 . PMC 102836 (free full text).
  21. ↑ Rand K, Qu W, Ho T, Clark SJ, Molloy P: Conversion-specific detection of DNA methylation using real-time polymerase chain reaction (ConLight-MSP) to avoid false positives . In: Methods . 27, No. 2, June 2002, pp. 114-20. doi : 10.1016 / S1046-2023 (02) 00062-2 . PMID 12095268 .
  22. ↑ Akey DT, Akey JM, Zhang K, Jin L: Assaying DNA methylation based on high-throughput melting curve approaches . In: Genomics . 80, No. 4, October 2002, pp. 376-84. doi : 10.1006 / geno.2002.6851 . PMID 12376091 .
  23. ↑ Kristensen LS, Mikeska T, Krypuy M, Dobrovic A: Sensitive Melting Analysis after Real Time- Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection . In: Nucleic Acids Res . 36, No. 7, April 2008, p. E42. doi : 10.1093 / nar / gkn113 . PMID 18344521 . PMC 2367707 (free full text).
  24. ↑ Adorján P, Distler J, Lipscher E, et al. : Tumor class prediction and discovery by microarray-based DNA methylation analysis . In: Nucleic Acids Res . 30, No. 5, March 2002, p. E21. doi : 10.1093 / nar / 30.5.e21 . PMID 11861926 . PMC 101257 (free full text).