Gene-Specific DNA Methylation - PowerPoint Presentation

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Gene-Specific DNA MethylationDetection Methods

Daniel GoanAnna TsengJoanna TychowskiFeb 2, 2012Slide2

MSP

Overview

DNA Digestion Based

COBRA

Real-time MSP

MethyLight

Bisulfite Sequencing

Pyrosequencing

MassARRAY

Southern-blot hybridization

Bisulfite BasedSlide3

Southern Blot Hybridization1978Slide4
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Southern Blot Hybridization

1978Slide7

Bisulfite Sequencing1992Slide8
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Bisulfite SequencingSlide12

COBRA/ Bio-COBRA(Combined Bisulfite Restriction

Analysis)Slide13
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COBRA/ Bio-COBRA(Combined Bisulfite Restriction

Analysis)Slide16

COBRA/ Bio-COBRA

(Combined Bisulfite Restriction

Analysis)Slide17
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PCR Needs:Template DNAPolymerasePrimers (Forward + Reverse)dNTPsBuffer

PCR ReviewSlide19

Methylation-Specific PCR Primer to methylated or unmethylated sequences

Real-time MSPMethyLightSYBR Green 1TaqMan*All 3 start with Bisulfite treatmentSlide20

C

U

1. Bisulfite treatment

2. Methylation specific primers/

Non-methylation specific primers

+

RNAP extends primers

Methylation Specific PCR

C

G

C

G

CH

3

C

G

CH

3

G

C

UGTC

Test for presence/absence of methylation

Primer:

(Herman, 1996)Slide21

C

G

C

G

C

G

T

T

G

G

T

G

CH

3

CH

3

CH

3

3. Gene-specific

PCR

Amplification

4.Sequencing

Look for T/C

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

Methylation Specific PCRSlide22

Methylation Specific PCRSmall amount of DNA

Easy to useLow cost-Qualitative -Presence/absence of meth/unmeth DNA moleculesProsConsSlide23

Real-time MSP MethyLight

Quenched

TaqMan

Fluorescing

TaqMan

C

G

C

C

CH

3

CH

3

CH

3

G

C

G

G

3’Quencher

5’Fluorophore

Bisulfite treatment

Meth specific primer

Bind

TaqMan probeRun PCR w/TaqMan PolMeasure fluorescence

(Eads C, 2000)Slide24

Real-time MSP MethyLight

-Quantitative-Sequence specific-Small amount of DNA-Easy to use -Expensive-Requires probe-One meth patternProsConsSlide25

Real-time MSP SYBR Green

SYBR Green (prefers GC-rich)

Add meth specific primer

Add dye

Run PCR

Dye binds DS

DNA+fluorescence

Measure fluorescence

C

C

CH

3

CH

3

(Hatterman

, 2008)Slide26

Real-time MSP SYBR Green -Quantitative-GC

specific-Small amount of DNA-Easy to use -Less expensive than TaqMan-Not as specific as TaqManProsConsSlide27

Proceed with Caution

Primer Design -Need methylated and unmethylated primersPCR Cycles-Optimum number of PCR cyclesTemp-Fully methylated DNA (CG-rich)-Unmethylated DNA (TG-rich)Dyes-Don’t inhibit PCR(Tollefsbol, Chapter 8)Slide28

MethyLight in Cervical Cancer DiagnosisLow Grade Lesions

High Grade LesionsCIN1CIN2CIN3

(Cancer)

Check Methylation Patterns!Slide29

Methylation Patterns can Distinguish Lesion Grades

SpecificSensitivePotential for high-throughput(Lim E, et al. 2010)CCNAI PAX1 DAPK1 TFI2 HS3ST2Slide30

Pyrosequencing

DNA templateDNA polymereasePrimerdNTPATP sulfurylaseLuciferaseLuciferinApyraseAPS(Adenosine 5’

phosphosulfate

)

APS

Sulfurylase

ATP

Luciferase

Luciferin

oxyluciferin

Light

dNTP

, dNMP, Phosphate

ATPApyrasedNTPADP, 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

Time

G

TTSlide31

Pyrosequencing Animationhttp://www.pyrosequencing.com/DynPage.aspx?id=7454

http://www.youtube.com/watch?v=kYAGFrbGl6ESlide32

Characteristics of PyrosequencingSmall amount of DNA neededHigh accuracy and flexibility in selecting gene of interest

Give quantitative dataEasy to use sofeware available Require design of suitable primerHigh costSlide33

Hypomethylation of

retrotransposable elements correlates with genomic instability in non‐small cell lung cancerInternational 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#fig1LINE-1; Normal

LINE-1; Lung Cancer

Alu

; Normal

Alu

; Lung CancerSlide34

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

Bisulfite TreatmentMethylated DNAUnmethylated DNACmG

C G

PCR

C

G

U G

In vitro

Transcription with T7 Polymerase

C G

T7 Primer

T G

T7 Primer

Base-specific RNA Cleavage

T7

G C

T7

A C

RNase

A

RNase

A

Data from

MALDI-TOF-MS

GC

ACSlide35

Characteristics of Mass-ArrayOnly a small amount of DNA needed High flexibility in selecting gene of interestGive quantitative dataAble to quantify large amount of sample (

upto 6000bp in one reactionPrimer 7 works for both methylated and methylated regionCostly equipment Slide36

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)Slide37

Technique

QuantitativeQualitativeSouthern-blot hybridization XBisulfite sequencing XCOBRA XMSP XReal-time MSP

X

Pyrosequencing

X

MassARRAY

XSlide38

ReferencesDaskalos, 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.23849Jun 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: 62-71Eads, 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 Slide39

ReferencesSlide40