Gene-Specific DNA Methylation Detection Methods

Daniel Goan Anna Tseng Joanna Tychowski Feb 2, 2012

Overview

Southern-blot hybridization DNA Digestion Based Bisulfite Sequencing COBRA 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(1978), not as widely used any more due to the development of PCR and other cheap/fast sequencing techniques, not outdated but just not as widely employed Process: • Step 1 use restriction enzymes to cut DNA • Step 2 electrophoresis to separate DNA fragments by size • • • Step 3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 SYBR Green 1

Real-time MSP

*All 3 start with Bisulfite treatment

MethyLight

TaqMan

CH 3

C

Methylation Specific PCR

Test for presence/absence of methylation

G

CH 3

C G G C

C  U

C G U G T C

1. Bisulfite treatment Primer: 2. Methylation specific primers/ Non-methylation specific primers + RNAP extends primers (Herman, 1996)

Methylation Specific PCR

CH 3

C G T G

3. Gene-specific PCR CH 3

C

Amplification CH 3

C G G T G T G

4.Sequencing

Look for T/C http://www.youtu

be.com/watch?v= zgNsmY6Led4

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 MethyLight

Quenched TaqMan 5’Fluorophore CH 3

C G G C

CH 3

C G

CH 3

C G

3’Quencher Fluorescing TaqMan 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

Pros -Quantitative -Sequence specific -Small amount of DNA -Easy to use Cons -Expensive -Requires probe -One meth pattern

Real-time MSP SYBR Green

C

CH 3

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 -Quantitative -GC specific -Small amount of DNA -Easy to use -Less expensive than TaqMan Cons -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 High Grade Lesions CIN1 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 oxyluciferin Luciferin G

P P P

dNTP

Pi Pi

Sulfurylase APS dNTP, dNMP, Phosphate ATP Luciferase 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

Bisulfite Treatment Methylated DNA Unmethylated DNA C G C m G C G U G PCR

In vitro

Transcription with T7 Polymerase C G T G

T7 Primer T7 Primer

Base-specific RNA Cleavage

RNase A

G C

T7

A C

RNase A T7

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

Southern-blot hybridization Bisulfite sequencing COBRA MSP Real-time MSP

Quantitative

X Pyrosequencing MassARRAY X X

Qualitative

X X 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 0006-291X, 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 O

6

-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

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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 o u

, . 2010 Feb;29(2):163-6.

Promoter methylation of CHFR gene in gastric carcinom a tissues detected using two methods.

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 o e

2011 Nov 4. [Epub ahead of print]

u

, Ferguson S , Gautrey HE , van Otterdijk SD , Hili M , Rand V , Moorman A , Meyer S , Brow n R , Strathdee G .

New castle, UK;

r S c o

2010 Jun;23(6):1675-82.

e

, Jeong D , Kim J , Yi L , Koo K , Lee J , Lee SD , Park JW , Chang B , Kim CH , Kim CJ , Lee MS .

u

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