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

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

3. Southern Blot Hybridization 1978

6. Southern Blot Hybridization 1978

7. Bisulfite Sequencing 1992

11. Bisulfite Sequencing

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

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

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

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

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

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

21. Methylation Specific PCR
CH3 C G T G
CH3 T G C G
3. Gene-specific
PCR
Amplification
CH3 T G C G
4.Sequencing
Look for T/C

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

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

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

25. 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)

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

27. Proceed with Caution Primer Design PCR Cycles Temp Dyes
-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)

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

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

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

31. Pyrosequencing Animation

32. 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

33. 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
5’Prompter CpG island of LINE-1
Pyrogram 3’ end CpG island of alu element
Representative pyrograms of the LINE‐1 and Alu sequences examined in our study. (a) LINE‐1; adjacent normal, (b) LINE‐1; lung tumor, (c) Alu; adjacent normal, (d) Alu; lung tumor. The percentages in boxes indicate the individual CpGs methylation values. The methylation index (MtI) is calculated as the average values of the examined CpGs.
© This slide is made available for non-commercial use only. Please note that permission may be required for re-use of images in which the copyright is owned by a third party.
International Journal of Cancer Volume 124, Issue 1, pages 81-87, 29 SEP 2008 DOI: /ijc

34. In vitro Transcription with T7 Polymerase Base-specific RNA Cleavage
Mass-Array Workflow
Bisulfite Treatment
Methylated DNA
Unmethylated DNA
CmG
C G
PCR
C G
U G
In vitro Transcription with T7 Polymerase
C G
T7 Primer
T G
Base-specific RNA Cleavage T7
G C
A C
RNase A
Data from
MALDI-TOF-MS GC AC
Adapted from:

35. 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

36. 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 (

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

38. 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:  /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 , ISSN X, /j.bbrc (
Quantative Methylation Analysis;
Principle of Pyrosequencing technology;
*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 CpG islands.
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):
Current protocols in protein science [ ] Brown, T yr:2001 vol:Appendix 4 pg:Appendix 4G -Appendix 4G
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 OneFile. Web. 29 Jan
A combined bisulfite restriction analysis bioinformatics tool: methyl-typing.
Methods in molecular biology [ ] Yang, Cheng-Hong yr:2011 vol:791 pg:73 -88

39. References