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
MassARRAY
Pyrosequencing
MSP
Real-time MSP
MethyLight
Southern Blot Hybridization
1978
-One of the older techniques for sequencing (1978), not as widely used any more due to the
development of PCR and other cheap/fast sequencing techniques, not outdated but just not as
widely employed
Process:
• Step 1 use restriction enzymes to cut DNA
• Step 2 electrophoresis to separate DNA fragments by size
• Step 3 transfer DNA fragments from gel to a membrane
• Step 4 hybridize radiolabeled probe to membrane
• Step 5 sample is washed to remove extraneous radio probes
Southern Blot Hybridization
1978
Advantages:
• Cheap
• simple
• 1 probe can detect multiple similar sequences
• well suited for analyzing long stretches of DNA
Disadvantages
• Requires a large amount of DNA
• Time consuming
• labor intensive
• limited sensitivity
Interesting probe gif
http://www.slic2.wsu.edu:82/hurlbert/micro101/images/101SouthernBlot10.gif
Bisulfite Sequencing
1992
-Treatment of DNA with sodium bisulfate to convert non methylated cytosines into uracil and
leaving 5-methylcytosine untouched in order to determine methylation percentage
Process:
• Step 1 melt (denature) DNA to separate strands
• Step 2 treat with sodium bisulfate
• Step 3 amplify converted DNA with PCR
• Step 4 Clone PCR product
• Step 5 Sequence
Bisulfite Sequencing
Advantages
• Accurate
• Does not require a lot of DNA
• cheap
• simple
Disadvantages
• possible DNA degradation/damage in chemical treatment
• possible incomplete conversion of DNA
• labor intensive
COBRA/ Bio-COBRA
(Combined Bisulfite Restriction Analysis)
-Builds upon bisulfite restriction analysis with the introduction of restriction enzymes and
polyacrylamide electrophoresis
Process:
• Step 1 melt (denature) DNA to separate strands
• Step 2 treat with sodium bisulfate
• Step 3 amplify converted DNA with PCR
• Step 4 Clone PCR product
• Step 5 treat PCR product with restriction enzymes (cleaves only methylated CpG's) (BstU1)
• Step 6 fragments separated by polyacrylamide gel electrophoresis
• Step 7 determine methylation percentage
COBRA/ Bio-COBRA
(Combined Bisulfite Restriction Analysis)
Advantages:
• simple
• quick
• cheap
• needs only small amount of DNA
• methylation percentages can be quantified
Disadvantages
• limited to restriction targets
• requires total chemical modification of DNA
COBRA/ Bio-COBRA
(Combined Bisulfite Restriction Analysis)
Bio COBRA: a variation of COBRA in which the
electrophoresis step is conducted in microfluidics chips,
allowing for the use of small amounts of liquid in the systems.
• Very sensitive
• Extremely fast
• Accurate
• Quantitative
• Small footprint
COBRA and Bio COBRA are both extremely useful in the detection and screening of methylation
states of biomarkers for cancer.
• TLX3 methylation in bladder cancer
• TWIST2 inactivation in leukemia
• hLHX6-HMR methylation in cervical cancer
• CHFR methylation in gastric cancer
PCR Review
PCR Needs:
Template DNA
Polymerase
Primers (Forward + Reverse)
dNTPs
Buffer
Methylation-Specific PCR
Primer to methylated or unmethylated sequences
TaqMan
SYBR Green 1
Real-time MSP
MethyLight
*All 3 start with Bisulfite treatment
Methylation Specific PCR
Test for presence/absence of methylation
CH3
C
G
C U
C
G
1. Bisulfite treatment
Primer:
CH3
C G
G C
UG
T C
2. Methylation specific primers/
Non-methylation specific primers
+
RNAP extends primers
(Herman, 1996)
Methylation Specific PCR
CH3
C
3. Gene-specific
PCR
Amplification
CH3
C
CH3
C
4.Sequencing
Look for T/C
G
T
G
G
T
G
G
T
G
http://www.youtu
be.com/watch?v=
zgNsmY6Led4
Methylation Specific PCR
Pros
Cons
-Qualitative
- Small amount of DNA
- Easy to use
- Low cost
-Presence/absence of meth/unmeth
DNA molecules
Real-time MSP MethyLight
3’Quencher
5’Fluorophore
Quenched TaqMan
Fluorescing TaqMan
CH3
C G
G C
1. Bisulfite treatment
2. Meth specific primer
3. Bind TaqMan probe
CH3
C
G
CH3
C
G
4. Run PCR w/TaqMan Pol
5. Measure fluorescence
(Eads C, 2000)
Real-time MSP MethyLight
Pros
Cons
-Quantitative
-Expensive
-Sequence specific
-Requires probe
-Small amount of DNA
-Easy to use
-One meth pattern
Real-time MSP SYBR Green
C
CH3
C
CH3
(Hatterman, 2008)
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
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
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)
MethyLight in Cervical Cancer
Diagnosis
Low Grade Lesions
CIN1
High Grade Lesions
CIN2
CIN3
(Cancer)
Check Methylation Patterns!
Methylation Patterns can Distinguish
Lesion Grades
CCNAI PAX1 DAPK1 TFI2 HS3ST2
Specific
Sensitive
Potential for high-throughput
(Lim E, et al. 2010)
Pyrosequencing
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
P
P
oxyluciferin
Pi Pi Sulfurylase
Luciferin
Luciferase
ATP
APS
dNTP, dNMP,
Phosphate
dNTP
Apyrase
ATP
ADP, AMP,
Phosphate
G TT
Time
Pyrosequencing Animation
• http://www.pyrosequencing.com/DynPage.as
px?id=7454
• http://www.youtube.com/watch?v=kYAGFrbGl
6E
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#fig1
Mass-Array Workflow
Methylated DNA
Unmethylated
DNA
C G
Bisulfite Treatment
CmG
PCR
In vitro
Transcription with
T7 Polymerase
C G
U G
C G
T G
T7 Primer
Base-specific
RNA Cleavage
G C
T7 Primer
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/
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
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)
Technique
Quantitative
Qualitative
Southern-blot
hybridization
X
Bisulfite
sequencing
X
COBRA
X
MSP
X
Real-time MSP
X
Pyrosequencing
X
MassARRAY
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 0006-291X,
10.1016/j.bbrc.2008.09.044. (http://www.sciencedirect.com/science/article/pii/S0006291X08017750)
•
Quantative Methylation 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 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): 225-231.
•
Current protocols in protein science [1934-3655] 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. 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
Additional sources for the cobra/bio cobra slide:
Int J Oncol. 2011 Sep;39(3):727-33. doi: 10.3892/ijo.2011.1049. Epub 2011 May 23.
Aberrant DNA methylation of T-cell leukemia, homeobox 3 modulates cisplatin sensitivity in bladder cancer.
Tada Y, Yokomizo A, Shiota M, Tsunoda T, Plass C, Naito S.
Department of Urology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
S
Chin J Cancer. 2010 Feb;29(2):163-6.
o
Promoter methylation of CHFR gene in gastric carcinoma tissues detected using two methods.
Cheng
u ZD, Hu SL, Sun YB, Xu WP, Shen G, Kong XY.
Department of Gerontology, Province Hospital of Anhui Medical University, Hefei, Anhui 230001, PR China.
r
S
c
Haematologica.
2011 Nov 4. [Epub ahead of print]
o
Epigenetic inactivation of TWIST2 in acute lymphoblastic leukemia modulates proliferation, cell survival and chemosensitivity.
eThathia SH, Ferguson S, Gautrey HE, van Otterdijk SD, Hili M, Rand V, Moorman A, Meyer S, Brow n R, Strathdee G.
u
New castle, UK;
r
S
cOncol Rep. 2010 Jun;23(6):1675-82.
The
o role of hLHX6-HM R as a methylation biomarker for early diagnosis of cervical cancer.
Jung S, Jeong D, Kim J, Yi L, Koo K, Lee J, Lee SD, Park JW, Chang B, Kim CH, Kim CJ, Lee MS.
e
u
Division of Biological Science and Research Center for Women's Diseases, Sookmyung Women's University, Seoul 140-742, Korea.
S
r
o
c
u
e
r