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Gene-Specific DNA Methylation Detection Methods

Gene-Specific DNA Methylation Detection Methods. Daniel Goan Anna Tseng Joanna Tychowski Feb 2, 2012. Overview. Southern-blot hybridization. DNA Digestion Based. COBRA. MSP. Bisulfite Sequencing. Bisulfite Based. Real-time MSP MethyLight. MassARRAY. Pyrosequencing.

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Gene-Specific DNA Methylation Detection Methods

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  1. Gene-Specific DNA MethylationDetection Methods Daniel Goan Anna Tseng Joanna Tychowski Feb 2, 2012

  2. Overview Southern-blot hybridization DNA Digestion Based COBRA MSP Bisulfite Sequencing Bisulfite Based Real-time MSP MethyLight MassARRAY Pyrosequencing

  3. Southern Blot Hybridization1978

  4. Southern Blot Hybridization1978

  5. Bisulfite Sequencing1992

  6. Bisulfite Sequencing

  7. COBRA/ Bio-COBRA(Combined Bisulfite Restriction Analysis)

  8. COBRA/ Bio-COBRA(Combined Bisulfite Restriction Analysis)

  9. COBRA/ Bio-COBRA(Combined Bisulfite Restriction Analysis)

  10. PCR Review PCR Needs: Template DNA Polymerase Primers (Forward + Reverse) dNTPs Buffer

  11. Methylation-Specific PCR Primer to methylated or unmethylated sequences TaqMan SYBR Green 1 MethyLight Real-time MSP *All 3 start with Bisulfite treatment

  12. Methylation Specific PCR Test for presence/absence of methylation CH3 CH3 G C C G G C G C U U C G T C 1. Bisulfite treatment 2. Methylation specific primers/ Non-methylation specific primers + RNAP extends primers Primer: (Herman, 1996)

  13. Methylation Specific PCR CH3 C G G T G CH3 T C G 3. Gene-specific PCR Amplification G T CH3 C G http://www.youtube.com/watch?v=zgNsmY6Led4 4.Sequencing Look for T/C

  14. Methylation Specific PCR Pros Cons -Qualitative -Presence/absence of meth/unmeth DNA molecules • Small amount of DNA • Easy to use • Low cost

  15. Real-time MSP MethyLight Quenched TaqMan 3’Quencher 5’Fluorophore Fluorescing TaqMan CH3 C G C Bisulfite treatment Meth specific primer Bind TaqMan probe Run PCR w/TaqMan Pol Measure fluorescence G CH3 CH3 C C G G (Eads C, 2000)

  16. Real-time MSP MethyLight Cons Pros -Quantitative -Sequence specific -Small amount of DNA -Easy to use -Expensive -Requires probe -One meth pattern

  17. Real-time MSP SYBR Green CH3 CH3 C C SYBR Green (prefers GC-rich) Add meth specific primer Add dye Run PCR Dye binds DS DNA+fluorescence Measure fluorescence (Hatterman, 2008)

  18. Real-time MSP SYBR Green Cons Pros -Quantitative -GC specific -Small amount of DNA -Easy to use -Less expensive than TaqMan -Not as specific as TaqMan

  19. Proceed with Caution Primer Design -Need methylated and unmethylated primers PCR Cycles -Optimum number of PCR cycles Temp -Fully methylated DNA (CG-rich) -Unmethylated DNA (TG-rich) Dyes -Don’t inhibit PCR (Tollefsbol, Chapter 8)

  20. MethyLight in Cervical Cancer Diagnosis High Grade Lesions Low Grade Lesions CIN3 CIN1 CIN2 (Cancer) Check Methylation Patterns!

  21. Methylation Patterns can Distinguish Lesion Grades CCNAI PAX1 DAPK1 TFI2 HS3ST2 Specific Sensitive Potential for high-throughput (Lim E, et al. 2010)

  22. Pyrosequencing DNA templateDNA polymerease Primer dNTP ATP sulfurylase LuciferaseLuciferin ApyraseAPS (Adenosine 5’ phosphosulfate) A G C C A A G G A A A C T C G G DNA Polymerase G P P P Pi Pi Sulfurylase ATP APS dNTP, dNMP, Phosphate oxyluciferin dNTP Luciferase Apyrase Light Luciferin ADP, AMP, Phosphate G TT ATP Time

  23. Pyrosequencing Animation • http://www.pyrosequencing.com/DynPage.aspx?id=7454 • http://www.youtube.com/watch?v=kYAGFrbGl6E

  24. Characteristics of Pyrosequencing • Small amount of DNA needed • High accuracy and flexibility in selecting gene of interest • Give quantitative data • Easy to use sofeware available • Require design of suitable primer • High cost

  25. Hypomethylation of retrotransposable elements correlates with genomic instability in non‐small cell lung cancer LINE-1; Normal Alu; Normal LINE-1; Lung Cancer Alu; Lung Cancer International Journal of CancerVolume 124, Issue 1, pages 81-87, 29 SEP 2008 DOI: 10.1002/ijc.23849http://onlinelibrary.wiley.com/doi/10.1002/ijc.23849/full#fig1

  26. Mass-Array Workflow MethylatedDNA UnmethylatedDNA Bisulfite Treatment C G CmG U G PCR C G In vitro Transcription with T7 Polymerase C G T G T7 Primer T7 Primer Base-specific RNA Cleavage G C A C T7 T7 RNase A RNase A GC AC Data from MALDI-TOF-MS Adapted from: http://www.sequenom.com/Files/Genetic-Analysis---Graphics/EpiTYPER---PDFs/Sequenom-Applications-Overview/

  27. Characteristics of Mass-Array • Only a small amount of DNA needed • High flexibility in selecting gene of interest • Give quantitative data • Able to quantify large amount of sample (upto 6000bp in one reaction • Primer 7 works for both methylated and methylated region • Costly equipment

  28. Quantitative analysis of human tissue-specific differences in methylation Jun Igarashi, et al. Quantitative analysis of human tissue-specific differences in methylation, Biochemical and Biophysical Research Communications, Volume 376, Issue 4, 28 November 2008, Pages 658-664 (http://www.sciencedirect.com/science/article/pii/S0006291X08017750)

  29. References • Daskalos, A., Nikolaidis, G., Xinarianos, G., Savvari, P., Cassidy, A., Zakopoulou, R., Kotsinas, A., Gorgoulis, V., Field, J. K. and Liloglou, T. (2009), Hypomethylation of retrotransposable elements correlates with genomic instability in non-small cell lung cancer. International Journal of Cancer, 124: 81–87. doi: 10.1002/ijc.23849 • Jun Igarashi, Satomi Muroi, Hiroyuki Kawashima, Xiaofei Wang, YuiShinojima, Eiko Kitamura, ToshinoriOinuma, NorimichiNemoto, Fei Song, SrimoyeeGhosh, William A. Held, Hiroki Nagase, Quantitative analysis of human tissue-specific differences in methylation, Biochemical and Biophysical Research Communications, Volume 376, Issue 4, 28 November 2008, Pages 658-664, ISSN 0006-291X, 10.1016/j.bbrc.2008.09.044. (http://www.sciencedirect.com/science/article/pii/S0006291X08017750) • QuantativeMethylation Analysis; http://www.sequenom.com/Files/Genetic-Analysis---Graphics/EpiTYPER---PDFs/Sequenom-Applications-Overview/ • Principle of Pyrosequencing technology; http://www.pyrosequencing.com/DynPage.aspx?id=7454 *Hattermann, K. et all (2008). A methylation-specific and SYBR-green-based quantitative polymerase chain reaction technique for O6-methylguanine DNA methyltransferase promoter methylation analysis. Analytical Biochemistry: (377)1: 62-71 • Eads, C. et al. (2000). MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Research: 28(8) • Herman, JG. Et al. (1996). Methylation-specific PCR: a novel PCR assay for methylation status of CpGislands. • Lime, E. et al. (2010) . Cervical dysplasia: assessing methylation status (Methylight) of CCNA1, DAPK1, HS3ST2, PAX1 and TFPI2 to improve diagnostic accuracy.Gynecologic Oncology. 119(2): 225-231. • Current protocols in protein science [1934-3655] Brown, T yr:2001 vol:Appendix 4 pg:Appendix 4G -Appendix 4G • Hansen, LiseLotte, et al. "Limitations and advantages of MS-HRM and bisulfite sequencing for single locus methylation studies." Expert Review of Molecular Diagnostics 10.5 (2010): 575+. Academic OneFile. Web. 29 Jan. 2012. • A combined bisulfite restriction analysis bioinformatics tool: methyl-typing. • Methods in molecular biology [1064-3745] Yang, Cheng-Hong yr:2011 vol:791 pg:73 -88

  30. References

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