Gene-specific Methylation Analyses

Fade Gong & Nam Nguyen

IntroductionDNA methylation in mammals occurs at CpG sites, where it serves many functions:

• Self vs. non-self identification (such as in viral/bacterial infection)

• Target for further regulatory events; examples:• Maintenance methyltransferases (functions after replication on hemi-methylated

DNA)• Proteins involved in chromatin folding

• CpG islands are elements of most promoters, which typically downregulate transcription in the methylated state

Principles of DNA Methylation Analyses

1.Single-gene methylation analyses

2. Genome-wide methylation analysesa. Anti-methylcytidine Abb. HPLC (high-performance liquid chromatography)/mass

spectrometry

a. Sensitivity of restriction enzymes for methylated CpG sites

b. Bisulfite-mediated conversion of DNA

Overview: Single-gene methylation analysesa) Methylation-sensitive restriction

enzymes• Southern-blot hybridization

b) Bisulfite conversion of DNA• Sequencing• Methylation-specific PCR (MSP)• Real-time MSP• COBRA• Pyrosequencing• MassARRAY

Southern-blot HybridizationRestriction endonucleases: CpG-sensitive• the majority are active on CpG and are inhibited by presence of

CMepG (we will focus on such an example: HpaII)

(Bird & Tollefsbol)

Southern-blot Hybridization (cont’d)Steps for Southern blotting:

1. Run DNA sample on gela. treated with HpaII, in our

exampleb. run against untreated

control (as in lanes 3 and 4, here)

2. Transfer to sticky membrane, such as nitrocellulose

3. Probe with oligonucleotide, conjugated to radioactive label

4. Expose to x-ray film

(Bird)

Southern-blot Hybridization (cont’d)

Advantages1. Relatively quantitative2. Low cost

Disadvantages3. Low flexibility in selection of DNA region (i.e. must be

isolated via other restriction enzymes that don’t degrade the fragment of interest)

4. Large amount of high-quality DNA required5. Not all CpG’s presented are subjected to the assay

(depends on selectivity of the restriction enzymes)

Polymerase Chain Reaction (PCR)

Review:

(McGraw-Hill Companies)

Bisulfite-mediated Conversion of DNA

Bisulfite Sequencing

PCR products are then sequenced and compared to the original sequence. CpG sites that remained CpG represent the presence of a 5-methylcytosine

Advantages:1. Provides detailed information about all CpG sites2. Requires smaller sample of DNA

Disadvantages:1. Labor-intensive

Combined Bisulfite Restriction Analysis (COBRA)

COBRA is based on the appearance or disappearance of a restriction site after bisulfite conversion

(Images are from wiki page of combined bisulfite restriction analysis)

Characteristics:Advantage: quantification; small amount DNA sample; cheapDisadvantage: CpG site regions are limited (Specific restriction site)

Methylation-specific PCR (MSP)

(Image was redrawn from the wiki page of Bisulfite sequencing)

• MSP takes advantage of changes in DNA sequence caused by bisulfite treatment

• Primers are designed that contain CpG sites and can thus select for expansion of methylated or unmethyled CpG’s

MSP (cont’d)

Advantages:• Simple technique• Flexibility• Cheap

Disadvantages: • Optimal number of PCR

cycles & annealing temperatures

• Appropriate negative control• Not a quantification method

Real-time MSPOverview: quantitative PCR (qPCR)/ Real-time PCR

• Double-stranded DNA-binding dyes as reporters (SYBR Green)

• Fluorescent reporter probe method (MethyLight: a Taqman probe)

(Image from Life technology website)

Real-time MSP (cont’d)

Typical amplification plot for qPCR Comparison between qPCR and PCR

(Images are from Qiagen website)

Real-time MSP (cont’d)Experiment Procedure: Bisulfite conversion; design methylation- and unmethylation-specific primers

Results analysis: Comparing amplification of test samples with standard samples that contain known numbers of DNA molecules.

Characteristics:High flexibility; accuracy; quantification; small amount samples; low cost

Methylight has higher specificity than SYBR Green based real-time MSP

Pyrosequencing

(Nature methods, 2005)

Overview:

((PPi = pyrophosphate!))

(Nature methods, 2005)

Pyrosequencing for CpG Methylation

CharacteristicsAdvantage:• Small amount of DNA• Flexibility• Accuracy• Quantification, etc.

Disadvantage:• Suitable primers• Expensive• Specific instruments

MassARRAY

Procedure: Bisulfite conversionPCRIn vitro transcriptionRNase digestionMass Spectrometry

Readout:Mass of products withC or T (16Da)

Base Specific RNasesRNase A: digest pyrimidine (Uracil)

MassARRAY

(SEQUENOM, Product Preview Note, 2005)

MassARRAYCharacteristics:

Advantage: • Small amount of

DNA• Flexibility• Quantification

Disadvantage:• Expensive• Specific instruments

ReferencesBird, A. P., Southern, E. M. “Use of Restriction Enzymes to Study Eukaryotic DNA Methylation” . J. Mol. Biol. 118 (1978). 27-37.

Critical Factors for Successful Real-Time PCR. Qiagen, Real-Time PCR Brochure, 2010.

Ehrich, M. et al., Introduction to DNA Methylation Analysis Using the MassARRAY System. SEQUENOM Preview Note, 2005.

Herman, James G. “Methylation-specific PCR: A novel PCR assay for methylation status of CpG islands”. Proc, Natl. Acad. Sci. USA. 93 (1996). 9821-9826.

Pyro Q-CpGTM: quantitative analysis of methylation in multiple CpG sites by Pyrosequencing. Nature Methods 2005, 2. doi:10.1038/nmeth800

TaqMan® Chemistry vs. SYBR® Chemistry for Real-Time PCR. Life Technologies, qPCR Education.

Tollefsbol, Trygve. Handbook of Epigenetics: The New Molecular and Medical Genetics. Oxford, UK: Elsevier Inc., 2011. 125-134. Print.

Wikipedia: Bisulfite sequencing http://en.wikipedia.org/wiki/Bisulfite_sequencing

Wikipedia: Combined bisulfite restriction analysishttp://en.wikipedia.org/wiki/Combined_bisulfite_restriction_analysis