Gene-specific Methylation Analyses
Fade Gong & Nam Nguyen
Introduction
DNA 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
a.
b.
Sensitivity of restriction
enzymes for methylated
CpG sites
Bisulfite-mediated
conversion of DNA
2. Genome-wide methylation analyses
a. Anti-methylcytidine Ab
b. HPLC (high-performance liquid chromatography)/mass spectrometry
Overview: Single-gene methylation analyses
a) Methylation-sensitive restriction enzymes
•
Southern-blot hybridization
b) Bisulfite conversion of DNA
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•
Sequencing
Methylation-specific PCR (MSP)
Real-time MSP
COBRA
Pyrosequencing
MassARRAY
Southern-blot Hybridization
Restriction 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 gel
a. treated with HpaII, in our
example
b. 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)
Advantages
1. Relatively quantitative
2. Low cost
Disadvantages
1. Low flexibility in selection of DNA region (i.e. must be
isolated via other restriction enzymes that don’t degrade the
fragment of interest)
2. Large amount of high-quality DNA required
3. 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 sites
2. 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; cheap
Disadvantage: CpG site regions are limited (Specific restriction site)
Methylation-specific PCR (MSP)
• 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
(Image was redrawn from
the wiki page of Bisulfite
sequencing)
MSP (cont’d)
Advantages:
Disadvantages:
• Simple technique
• Flexibility
• Cheap
• Optimal number of PCR
cycles & annealing
temperatures
• Appropriate negative control
• Not a quantification method
Real-time MSP
Overview: 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
Overview:
((PPi = pyrophosphate!))
(Nature methods, 2005)
Pyrosequencing for
CpG Methylation
Characteristics
Advantage:
• Small amount of DNA
• Flexibility
• Accuracy
• Quantification, etc.
Disadvantage:
• Suitable primers
• Expensive
• Specific instruments
(Nature methods, 2005)
MassARRAY
MassARRAY
Procedure:
Bisulfite conversion
PCR
In vitro transcription
RNase digestion
Mass Spectrometry
Readout:
Mass of products with
C or T (16Da)
Base Specific RNases
RNase A:
digest pyrimidine (Uracil)
(SEQUENOM, Product Preview Note, 2005)
MassARRAY
Characteristics:
Advantage:
• Small amount of DNA
• Flexibility
• Quantification
Disadvantage:
• Expensive
• Specific instruments
References
Bird, 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 analysis
http://en.wikipedia.org/wiki/Combined_bisulfite_restriction_analysis