Gene-Specific DNA MethylationDetection Methods

Daniel GoanAnna Tseng

Joanna Tychowski

Feb 2, 2012

MSP

Overview

DNA Digestion Based

COBRA

Real-time MSP MethyLight

Bisulfite Sequencing

Pyrosequencing

MassARRAY

Southern-blot hybridization

Bisulfite Based

Southern Blot Hybridization1978

-One of the older techniques for sequencing (1978), not as widely used any more due to thedevelopment of PCR and other cheap/fast sequencing techniques, not outdated but just not aswidely employed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

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

Southern Blot Hybridization1978

Interesting probe gif http://www.slic2.wsu.edu:82/hurlbert/micro101/images/101SouthernBlot10.gif

Bisulfite Sequencing1992

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

-Treatment of DNA with sodium bisulfate to convert non methylated cytosines into uracil andleaving 5-methylcytosine untouched in order to determine methylation percentage

Advantages• Accurate • Does not require a lot of DNA• cheap • simple

Bisulfite Sequencing

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 andpolyacrylamide 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 theelectrophoresis 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 methylationstates 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 Needs:Template DNAPolymerasePrimers (Forward + Reverse)dNTPsBuffer

PCR Review

Methylation-Specific PCR Primer to methylated or unmethylated sequences

Real-time MSPMethyLight

SYBR Green 1TaqMan

*All 3 start with Bisulfite treatment

C U

1. Bisulfite treatment 2. Methylation specific primers/Non-methylation specific primers + RNAP extends primers

Methylation Specific PCR

C G

C G

CH3

C GCH3

G C

U GT C

Test for presence/absence of methylation

Primer: (Herman, 1996)

C G

C G

C G

T

T

G

G

T GCH3

CH3

CH3

3. Gene-specific PCR Amplification

4.SequencingLook for T/C

http://www.youtube.com/watch?v=zgNsmY6Led4

Methylation Specific PCR

Methylation Specific PCR

- Small amount of DNA

- Easy to use

- Low cost

-Qualitative -Presence/absence of meth/unmeth DNA molecules

Pros Cons

Real-time MSP MethyLightQuenched TaqMan

Fluorescing TaqMan

C G

C C

CH3

CH3 CH3

G C

G G

3’Quencher5’Fluorophore

1. Bisulfite treatment

2. Meth specific primer

3. Bind TaqMan probe

4. Run PCR w/TaqMan Pol

5. Measure fluorescence

(Eads C, 2000)

Real-time MSP MethyLight

-Quantitative

-Sequence specific

-Small amount of DNA

-Easy to use

-Expensive

-Requires probe

-One meth pattern

Pros Cons

Real-time MSP SYBR Green

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

C CCH3 CH3

(Hatterman, 2008)

Real-time MSP SYBR Green

-Quantitative

-GC specific

-Small amount of DNA

-Easy to use

-Less expensive than TaqMan

-Not as specific as TaqMan

Pros Cons

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 High Grade Lesions

CIN1 CIN2 CIN3(Cancer)

Check Methylation Patterns!

Methylation Patterns can Distinguish Lesion Grades

SpecificSensitivePotential for high-throughput

(Lim E, et al. 2010)

CCNAI PAX1 DAPK1 TFI2 HS3ST2

PyrosequencingDNA templateDNA polymereasePrimerdNTPATP sulfurylaseLuciferaseLuciferinApyraseAPS(Adenosine 5’ phosphosulfate )

APS

Sulfurylase

ATP

Luciferase

Luciferinoxyluciferin

Light

dNTP, dNMP, Phosphate

ATP

Apyrase

dNTP

ADP, AMP, Phosphate

A G C C A A G G A A A C T C G G

DNA Polymerase

G P P P Pi Pi

TimeG TT

Pyrosequencing Animation

• http://www.pyrosequencing.com/DynPage.aspx?id=7454

• http://www.youtube.com/watch?v=kYAGFrbGl6E

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‐

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

LINE-1; Normal

LINE-1; Lung Cancer

Alu; Normal

Alu; Lung Cancer

Mass-Array Workflow

Adapted from: http://www.sequenom.com/Files/Genetic-Analysis---Graphics/EpiTYPER---PDFs/Sequenom-Applications-Overview/

Bisulfite TreatmentMethylated DNA Unmethylated

DNACmG C G

PCR C G U G

In vitro Transcription with

T7 PolymeraseC G

T7 Primer

T G

T7 Primer

Base-specific RNA Cleavage T7G C T7A C

RNase A RNase A

Data fromMALDI-TOF-MS

GC AC

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

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.

Chin J Cancer . 2010 Feb;29(2):163-6.Promoter methylation of CHFR gene in gastric carcinoma tissues detected using two methods.Cheng 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.

Haematologica. 2011 Nov 4. [Epub ahead of print]Epigenetic inactivation of TWIST2 in acute lymphoblastic leukemia modulates proliferation, cell survival and chemosensitivity.Thathia SH, Ferguson S, Gautrey HE, van Otterdijk SD, Hili M, Rand V, Moorman A, Meyer S, Brow n R, Strathdee G.

New castle, UK;

Oncol Rep. 2010 Jun;23(6):1675-82.The role of hLHX6-HMR 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.

Division of Biological Science and Research Center for Women's Diseases, Sookmyung Women's University, Seoul 140-742, Korea.

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