GeneSpecific DNA Methylation Detection Methods Daniel Goan Anna

Gene-Specific DNA Methylation Detection Methods Daniel Goan Anna Tseng Joanna Tychowski Feb 2, 2012

Overview Southern‐blot hybridization DNA Digestion Based COBRA Bisulfite Sequencing Bisulfite Based Mass. ARRAY Pyrosequencing MSP Real‐time MSP Methy. Light

Southern Blot Hybridization 1978

Southern Blot Hybridization 1978

Bisulfite Sequencing 1992

Bisulfite Sequencing

COBRA/ Bio-COBRA (Combined Bisulfite Restriction Analysis)

COBRA/ Bio-COBRA (Combined Bisulfite Restriction Analysis)

COBRA/ Bio-COBRA (Combined Bisulfite Restriction Analysis)

PCR Review PCR Needs: Template DNA Polymerase Primers (Forward + Reverse) d. NTPs Buffer

Methylation-Specific PCR Primer to methylated or unmethylated sequences Taq. Man SYBR Green 1 Real‐time MSP Methy. Light *All 3 start with Bisulfite treatment

Methylation Specific PCR Test for presence/absence of methylation CH 3 C C U C G G 1. Bisulfite treatment Primer: CH 3 C G G C UG T C 2. Methylation specific primers/ Non-methylation specific primers + RNAP extends primers (Herman, 1996)

Methylation Specific PCR CH 3 C CH 3 3. Gene-specific PCR Amplification C CH 3 C 4. Sequencing Look for T/C G T G http: //www. youtu be. com/watch? v= zg. Nsm. Y 6 Led 4

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

Real-time MSP Methy. Light 3’Quencher 5’Fluorophore Quenched Taq. Man Fluorescing Taq. Man CH 3 C G G C 1. Bisulfite treatment 2. Meth specific primer 3. Bind Taq. Man probe CH 3 C G 4. Run PCR w/Taq. Man Pol 5. Measure fluorescence (Eads C, 2000)

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

Real-time MSP SYBR Green C CH 3 SYBR Green (prefers GC-rich) 1. Add meth specific primer 2. Add dye 3. Run PCR 4. Dye binds DS DNA+fluorescence 5. Measure fluorescence (Hatterman, 2008)

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

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)

Methy. Light in Cervical Cancer Diagnosis Low Grade Lesions CIN 1 High Grade Lesions CIN 2 CIN 3 (Cancer) Check Methylation Patterns!

Methylation Patterns can Distinguish Lesion Grades CCNAI PAX 1 DAPK 1 TFI 2 HS 3 ST 2 Specific Sensitive Potential for high-throughput (Lim E, et al. 2010)

Pyrosequencing DNA template A G C C A A G G A A A C DNA T C G G polymerease DNA Luciferin oxyluciferin Primer Polymerase d. NTP ATP sulfurylase G P Pi Pi Sulfurylase Luciferase Luciferin ATP APS Light Apyrase APS d. NTP, d. NMP, (Adenosine 5’ Phosphate d. NTP phosulfate ) Apyrase ATP ADP, AMP, Phosphate G TT Time

Pyrosequencing Animation • http: //www. pyrosequencing. com/Dyn. Page. as px? id=7454 • http: //www. youtube. com/watch? v=k. YAGFrb. G l 6 E

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

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 Cancer Volume 124, Issue 1, pages 81 -87, 29 SEP 2008 DOI: 10. 1002/ijc. 23849 http: //onlinelibrary. wiley. com/doi/10. 1002/ijc. 23849/full#fig 1

Mass‐Array Workflow Bisulfite Treatment Methylated DNA Unmethylated DNA C G Cm. G PCR In vitro Transcription with T 7 Polymerase C G U G C G T 7 Primer Base‐specific RNA Cleavage G C T 7 Primer A C T 7 RNase A Data from MALDI‐TOF‐MS T 7 GC AC Adapted from: http: //www. sequenom. com/Files/Genetic-Analysis---Graphics/Epi. TYPER---PDFs/Sequenom-Applications-

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 6000 bp in one reaction • Primer 7 works for both methylated and methylated region • Costly equipment • •

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/S 0006291 X 08017750 )

Technique Quantitative Qualitative Southern-blot hybridization X Bisulfite sequencing X COBRA X MSP X Real-time MSP Pyrosequencing Mass. ARRAY X X

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, Yui Shinojima, Eiko Kitamura, Toshinori Oinuma, Norimichi Nemoto, Fei Song, Srimoyee Ghosh, 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 -291 X, 10. 1016/j. bbrc. 2008. 09. 044. (http: //www. sciencedirect. com/science/article/pii/S 0006291 X 08017750) • Quantative Methylation Analysis; http: //www. sequenom. com/Files/Genetic-Analysis---Graphics/Epi. TYPER---PDFs/Sequenom-Applications-Overview/ • Principle of Pyrosequencing technology; http: //www. pyrosequencing. com/Dyn. Page. aspx? id=7454 *Hattermann, K. et all (2008). A methylation‐specific and SYBR‐green‐based quantitative polymerase chain reaction technique for O 6 -methylguanine DNA methyltransferase promoter methylation analysis. Analytical Biochemistry: (377)1: 62‐ 71 • Eads, C. et al. (2000). Methy. Light: 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 Cp. G islands. • Lime, E. et al. (2010). Cervical dysplasia: assessing methylation status (Methylight) of CCNA 1, DAPK 1, HS 3 ST 2, PAX 1 and TFPI 2 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 4 G -Appendix 4 G • Hansen, Lise Lotte, 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 One. File. 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

References