Biogerontology 4: 233–250, 2003.

© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

233

Methods

DNA methylation analysis techniques

Christina Dahl & Per Guldberg∗

Institute of Cancer Biology, Danish Cancer Society, Strandboulevarden 49, 2100 Copenhagen, Denmark; ∗ Author
for correspondence (e-mail: perg@cancer.dk; fax: +45-35257721)

Received 12 December 2002; accepted in revised form 10 March 2003

Key words: bisulfite, DNA methylation, 5-methylcytosine, PCR, review

Abstract
DNA methylation contributes to the control of gene expression and plays an essential role in cellular physiology.
Well-defined patterns of DNA methylation are established and fixed during embryonic development, and changes
in these patterns may be a contributing factor in developmental disorders, cancer and aging. Not least the possibility
of using DNA methylation as a marker for disease has created a strong need for techniques to detect and measure
DNA methylation. Different techniques provide information on DNA methylation at different levels, spanning
from genome-wide methylation content to methylation of single residues in specific genes. The limitations of
individual techniques strongly affect interpretation of data. In this review, we discuss some general themes in DNA
methylation analysis and outline the basic principles of current key techniques. We discuss the advantages and
disadvantages of these techniques, including potential artifacts and pitfalls, and suggest some overall guidelines
that may be instructive for a rational choice of methodology.

Abbreviations: COBRA – combined bisulfite restriction analysis; DHPLC – denaturing high-performance liquid
chromatography; DMH – differential methylation hybridization; HPCE – high-performance capillary electro-
phoresis; HPLC – high-performance liquid chromatography; MCA-RDA – methylated CpG island amplification-
representational difference analysis; MS-AP-PCR – methylation-sensitive arbitrarily primed PCR; MS-DGGE
– methylation-specific denaturing gradient gel electrophoresis; MS-DHPLC – methylation-specific denaturing
high-performance liquid chromatography; MS-MCA – methylation-specific melting curve analysis; Ms-SnuPE
– methylation-sensitive single nucleotide primer extension; MS-SSCA – methylation-specific single-strand
conformation analysis; MSO – methylation-specific microarray; MSRE – methylation-sensitive restriction
endonuclease; MSRF – methylation-sensitive restriction fingerprinting; PCR – polymerase chain reaction; RLGS
– restriction landmark genomic scanning; SAM – S-adenosylmethionine; TLC – thin-layer chromatography

Introduction methylated, i.e., cytosines within CpG dinucleotides.

Approximately 1% of the bases in mammalian
genomes are 5-methylcytosine. In contrast to the other
four bases in the genome, 5-methylcytosine does not
exist in the cellular environment as a free nucleotide.
Rather, the addition of a methyl group to cytosine
occurs after DNA replication and is catalyzed by
a group of DNA methyltransferase enzymes (Bestor
2000). The distribution of 5-methylcytosine is not ran-
dom in the genome. Notably, with few possible excep-
tions, only cytosines that precede guanines can be
Furthermore, while CpGs in bulk DNA are usually
methylated, methylation is absent in CpG-rich regions
(CpG islands) that are present near the 5 end of 50–
60% of all genes. Overall, ∼4% of cytosines and
∼75% of CpGs are methylated in genomic DNA
from normal human postnatal somatic tissues. The
biological significance of this particular distribution
of DNA methylation has become increasingly under-
stood through the unraveling of mechanisms by which
DNA methylation may contribute to the control of
gene transcription.
234
Methylation is the predominant ‘epigenetic’
modification of DNA in mammalian genomes. The
term epigenetic is used to denote that methylation
can modify the information content of DNA without
changing the primary nucleotide sequence. Hence,
DNA methylation does not alter the structure or
function of a gene, but provides information as to
where and when the gene should be expressed. There
is now substantial evidence that, in general, the
level of methylation within a gene’s promoter CpG
island correlates with its transcriptional silencing.
Altering the binding of some transcription factors is
one possible mechanism by which 5-methylcytosine
controls gene expression. An alternative model for
methylation-based gene silencing implicates a group
of proteins and protein complexes that specifically
bind to methyl-CpG and recruit histone deacetylates
to the sites of methylation (Wade 2001). The removal
of acetyl groups from the histones of the nucleosomal
core converts the open, transcriptionally competent
chromatin structure into a closed structure that is
inaccessible to the transcriptional machinery. A third
possible mechanism of methylation-dependent gene
silencing involves DNA methyltransferases, which
have transcriptional repressor function and can recruit
histone deacetylates to methylated DNA (Robertson
2002).
In humans, methylation-dependent gene silencing
has been implicated in inactivation of the X chromo-
some in females, inactivation of the silent allele at
imprinted loci, and silencing of intragenomic para-
sitic DNA elements (Costello and Plass 2001). Several
lines of research have shown that the establishment
and maintenance of well-defined methylation patterns
are required for normal embryonic development. First,
elimination of any of the three known DNA methyl-
transferases, Dnmt1, Dnmt3a and Dnmt3b, is lethal
in mice (Okano et al. 1999; Li et al. 1992). Second,
a number of human disorders are associated with
genetic abnormalities involving chromosomal regions
that are subject to genomic imprinting, includ-
ing Beckwith-Wiedemann syndrome, Prader-Willi
syndrome, and Angelman syndrome (Paulsen and
Ferguson-Smith 2001). Third, two human syndromes
have been identified that are caused by inherited
defects in genes affecting the DNA methylation
machinery. Rett syndrome, an X-linked, neuropsychi-
atric disorder with onset in early childhood, is caused
by loss-of-function mutations in the gene encoding
methyl-CpG binding protein MeCP2 (Amir et al.
1999), and mutations in the DNMT3B gene have been
found in ICF syndrome, a rare disorder characterized
by immunodeficiency, facial anomalies, and hypo-
methylation of the satellite DNA in juxtacentromeric
heterochromatin of chromosomes 1 and 16 (Okano et
al. 1999).
In normal, fully differentiated cells, DNA methyla-
tion patterns are replicated with high fidelity during
mitosis. However, a large body of data has shown that
these patterns can become altered and cause cellular
functional abnormalities in the course of human
disease and aging. The pathologic consequences of
altered DNA methylation have been most extensively
studied in cancer. Compared with normal cells, cancer
cells frequently show genome-wide hypomethylation,
hypermethylation of tumor suppressor genes, and
loss of genomic imprinting (Baylin et al. 1998).
Recent studies have demonstrated increased rates of
spontaneous mutations, deletions and rearrangements
in cells lacking DNMT1, the DNA methyltransferase
responsible for maintaining DNA methylation patterns
after DNA replication (Chen et al. 1998). Accord-
ingly, loss of genomic methylation may represent an
early event in multistep carcinogenesis by providing
the premalignant cell with a mutator phenotype. In
contrast, hypermethylation of CpG islands may cause
gene silencing and can be functionally equivalent to
mutation and deletion in activating tumor suppressor
genes (Jones and Laird 1999). Intriguingly, evidence is
emerging that many of the changes observed in cancer
cells are also present in aging cells in normal tissues. It
has been proposed that these age-related methylation
changes are precursors for the aberrant methylation
patterns in cancer cells and thus may contribute to
the age-related increase in cancer risk (Ahuja and Issa
2000).
The central role of DNA methylation in main-
taining cellular function and the recognition that
changes in DNA methylation patterns may have broad
implications for human health have created a strong
need for techniques to reliably detect and measure
methylation in DNA from diverse sources. In this
review, we describe different ways of analyzing DNA
methylation and outline the basic principles of current
techniques (listed in Table 1), with emphasis on tech-
niques for gene-specific analysis of DNA methylation.
Each of these techniques is limited in the information
content of the data it generates, which strongly affects
interpretation of data.
 235
Table 1. Techniques described in this review, grouped according to the dimensions in which they can resolve DNA methylation.

Initial treatment of PCR-

Application Technique genomic DNA based Key references

Analysis of genome- HPLC Enzymatic hydrolysis No Kuo et al. 1980

wide methylation HPCE Enzymatic hydrolysis No Fraga et al. 2002
content TLC Enzyme digestion No Schmitt et al. 1997
SssI acceptance assay None No Wu et al. 1993
Chloroacetaldehyde assay Bisulfite treatment No Oakeley et al. 1999
Immunochemical analysis Denaturation/depurination No Oakeley et al. 1997

Qualitative analysis MSRE-Southern Enzyme digestion No Southern 1975

of single CpGs MSRE-PCR Enzyme digestion Yes Singer-Sam et al. 1990
Methylation-specific PCR Bisulfite treatment Yes Herman et al. 1996
COBRA Bisulfite treatment Yes Xiong and Laird 1997
Direct bisulfite genomic sequencing Bisulfite treatment Yes Frommer et al. 1992
Cloned bisulfite genomic sequencing Bisulfite treatment Yes Frommer et al. 1992

Quantitative analysis Ms-SnuPE Bisulfite treatment Yes Gonzalgo and Jones 1997
of single CpGs MethyLight Bisulfite treatment Yes Eads et al. 2000
COBRA Bisulfite treatment Yes Xiong and Laird 1997
Direct bisulfite genomic sequencing Bisulfite treatment Yes Frommer et al. 1992

Analysis of allelic Direct bisulfite genomic sequencing Bisulfite treatment Yes Frommer et al. 1992
methylation Cloned bisulfite genomic sequencing Bisulfite treatment Yes Frommer et al. 1992
heterogeneity MethyLight Bisulfite treatment Yes Eads et al. 2000
MS-MCA Bisulfite treatment Yes Worm et al. 2001
MS-DGGE Bisulfite treatment Yes Aggerholm et al. 1999
MS-SSCA Bisulfite treatment Yes Maekawa et al. 1999
MS-DHPLC Bisulfite treatment Yes Baumer et al. 2001
MSO Bisulfite treatment Yes Gitan et al. 2002;
Adorjan et al. 2002

Random screening for RLGS Enzyme digestion No Costello et al. 2000

altered methylation MCA-RDA Enzyme digestion Yes Toyota et al. 1999
loci MS-AP-PCR Enzyme digestion Yes Gonzalgo et al. 1997
DMH Enzyme digestion Yes Huang et al. 1999

Analysis of genome-wide methylation content High-performance liquid chromatography (HPLC)

and related techniques
Data on genome-wide methylation content in a pool
of DNA is usually generated by determining the
For many years, HPLC has been the preferred tech-
ratio between 5-methylcytosine and cytosine. Current
nique for quantitative determination of the global
standard procedures involve complete enzymatic
levels of DNA methylation (Kuo et al. 1980). Accord-
hydrolysis of DNA, followed by high-resolution
ing to standard procedures, total genomic DNA is
separation to obtain the total base composition of the
hydrolyzed to deoxyribonucleotides (base + deoxyri-
genome. Alternative techniques have been developed,
bose + phosphate) using a combination of deoxyribo-
including assays based on the use of bacterial DNA
nuclease I and nuclease P1. Following hydrolysis,
methyltransferase or 5-methylcytosine specific anti-
deoxyribonucleotides are further converted into
bodies.
deoxyribonucleosides (base + deoxyribose) by treat-
ment with alkaline phosphatase, and the products
are then separated by standard reverse-phase HPLC.
236
Cytosine and 5-methylcytosine can be identified and
quantified by including external standards of bases
and monitoring UV absorbance at 254 nm. To avoid
interference from RNA contamination, it is usually
necessary to remove RNA from the nucleic acid
preparations by enzymatic hydrolysis using a combi-
nation of ribonuclease A and ribonuclease T1.
Fraga et al. (2002) used high-performance capil-
lary electrophoresis (HPCE) to resolve DNA hydroly-
sates and determine the content of 5-methylcytosine.
Compared with HPLC, HPCE is more simple, rapid
and cost-effective. The sensitivity of HPLC- or HPCE-
based determination of genome-wide DNA methyla-
tion is relatively high (<5 µg), but may still be too low
for routine processing of tissue specimens. Signifi-
cant improvements in sensitivity have been achieved
by combining HPLC separation of DNA hydrolysates
with mass spectrometry detection (del Gaudio et al.
1997), which also may be useful for HPCE-based
approaches.
In laboratories where equipment for high-resolu-
tion separation of hydrolyzed DNA is not available,
thin-layer chromatography (TLC) may be a useful
alternative (Schmitt et al. 1997). In brief, DNA is cut
at all CCGG sites using the restriction enzyme MspI,
and the internal cytosine in these sites is then radioac-
tively labeled using γ -labeled ATP and polynucleotide
kinase. Following hydrolysis of the DNA to mono-
nucleotides using nuclease P1, radioactively labeled
cytosine monophosphate and 5-methylcytosine mono-
phosphate are separated on TLC plates and identified
and quantified as individual spots using a phosphor-
imager. Because only cytosines in CCGG sites are
probed, this approach only provides a rough estimate
of the ratio between 5-methylcytosine and cytosine.
SssI acceptance assay
The bacterial enzyme SssI DNA methyltransferase,
which uses S-adenosylmethionine (SAM) as methyl
donor, effectively methylates unmethylated cytosine in
CpG sites. This enzyme can be used in a simple assay
to determine global CpG methylation: genomic DNA
is incubated with SssI methyltransferase in the pres-
ence of tritium-labeled SAM and then immobilized on
nitrocellulose paper (Wu et al. 1993). The amount of
radioactive label incorporated can be determined using
a scintillation counter and is a direct measure of the
number of unmethylated CpGs in the total pool of
DNA.
Despite its simplicity, the SssI acceptance assay is
prone to a number of technical problems. First of all,
the day-to-day variation is considerable, primarily due
to the instability of both SAM and SssI methyltrans-
ferase. Another problem may be associated with the
difficulty of dissolving genomic DNA to homogeneity,
which may lead to inaccurate DNA concentration
measurements. A useful way of obtaining a more
homogeneous solution is to digest the DNA with a
restriction enzyme (e.g., EcoRI) that does not contain
a CpG sequence in its recognition site (Oakeley 1999).
Chloroacetaldehyde assay
Oakeley et al. (1999) described a method for studying
changes in genome-wide levels of DNA methylation
by fluorescent labeling using chloroacetaldehyde. The
DNA is first treated with sodium bisulfite, which
converts cytosine into uracil, but leaves 5-methyl-
cytosine unchanged (Frommer et al. 1992; described
in detail below). The bisulfite-treated DNA is then
depurinated under acidic conditions, and the purines
are removed by silver nitrate precipitation or by
column chromatography. Subsequent incubation of the
sample with chloroacetaldehyde yields the intensely
fluorescent ethenocytosine derivative of 5-methyl-
cytosine, which can be quantified using a fluorimeter
and used as a direct measure of the level of 5-methyl-
cytosine in the genome. The chloroacetaldehyde assay
is an attractive alternative to the SssI acceptance assay.
Not least the use of stable and inexpensive chem-
icals and the avoidance of radioactivity are major
advantages.
Immunochemical analysis
Monoclonal antibodies raised against 5-methylcyto-
sine provide a powerful tool for studying global DNA
methylation. To determine genome-wide methylation
content, genomic DNA is denatured and immobil-
ized on a nitrocellulose membrane and then incubated
with an anti-5-methylcytosine antibody. The intensity
of the staining, which is proportional to the degree
of methylation, may be determined by subsequent
incubation with a fluorescein-conjugated secondary
antibody followed by fluorescence scanning (Oakeley
et al. 1997).
Immunofluorescence microscopy has been used to
map patterns of DNA methylation in cellular chromo-
somes in individual cells. Chromosomal regions
with particularly high densities of 5-methylcytosine,
such as juxtacentromeric regions, emit the strongest

fluorescence. A prerequisite for quantitative determi-
nation of DNA methylation using immunochemical
analysis is that 5-methylcytosine is not involved in
base pairing, which may be accomplished by depuri-
nating the DNA using sulphuric acid (Oakeley et al.
1997).
Basic principles of gene-specific methylation
analysis
While analysis of global DNA methylation content can
be determined directly on crude DNA preparations,
current procedures for analysis of gene-specific DNA
methylation patterns require initial amplification of the
target sequence. Because DNA polymerases do not
discriminate between cytosine and 5-methylcytosine,
all information on 5-methylcytosine will be erased
during polymerase chain reaction (PCR) and cloning
procedures. A number of enzymes and chemicals
have been used for covalent base modification to
allow discrimination between cytosine and 5-methyl-
cytosine, including methylation-sensitive restriction
endonucleases (MSREs), bisulfite (HSO− 3 ), hydrazine
−
(N2 H4 ) and permanganate (MnO4 ). All methods for
gene-specific methylation analysis described in this
review either use MSREs or bisulfite. For a more
comprehensive review of methods allowing identifica-
tion of 5-methylcytosine, see Rein et al. (1998).
MSREs
Isoschizomers of bacterial restriction endonucleases
with different sensitivities to 5-methylcytosine have
been widely used to determine the methylation status
of cytosine at specific sites. Usually, one of the
enzymes of the isoschizomer pair is insensitive to 5-
methylcytosine, while the other will not cut DNA if its
cleavage site contains a 5-methylcytosine (Figure 1A).
A popular isoschizomer pair for methylation analysis
is HpaII/MspI. Both enzymes cleave CCGG sites, but
HpaII is unable to cleave if the internal cytosine is
methylated.
Methods based on the use of MSREs generally
suffer from the limitation that they provide informa-
tion only about CpGs within the cleavage sites of the
specific enzymes. Furthermore, MSRE-based analysis
may be plagued by false positives because of incom-
plete digestion, resulting in the false conclusion that
at least some cytosines in the target sequence are
methylated.
237
The bisulfite method
Treatment of genomic DNA with sodium bisulfite can
effectively deaminate unmethylated cytosine residues
to uracil under conditions where 5-methylcytosine
is deaminated at a very slow rate (Frommer et al.
1992). Preferential deamination of cytosine by bisul-
fite occurs through sequential sulfonation, hydrolytic
deamination, and alkaline desulfonation reactions, and
leads to the conversion of the DNA double helix into
two single strands that are no longer complementary
(Figure 1B). According to the modification protocol
originally developed by Frommer et al. (1992),
genomic DNA is fragmented by shearing or diges-
tion with appropriate restriction enzymes, denatured
with sodium hydroxide, treated with bisulfite at pH
5, desalted and desulfonated with sodium hydroxide.
Finally, the DNA is neutralized, desalted and resolved
in water or storage buffer. The bisulfite-induced
sequence differences in DNA templates with different
contents of 5-methylcytosine form the basis for
discriminating between methylated and unmethylated
DNA. Bisulfite-treated DNA can be used directly
as a template in a standard PCR, in which all
uracil (formerly unmethylated cytosine) and thymine
residues will be amplified as thymine and only 5-
methylcytosine residues will be amplified as cytosine.
Compared with MSRE methods for analyzing
gene-specific methylation, methods relying on
bisulfite treatment have several advantages. Most
importantly, the methylation status of virtually any
CpG in the genome can be determined, both for
the total cell population and for individual DNA
molecules. The most severe limitation of the bisulfite
method is that it is highly prone to reaction artifacts,
which may affect its reliability. The most commonly
encountered artifact is incomplete conversion of
cytosine to uracil. Because bisulfite reacts only with
cytosines that are not involved in base pairing, the
most obvious explanation for incomplete conversion
of cytosine is incomplete denaturation of the DNA
template or its partial renaturation during bisulfite
treatment. Several modifications of the original
protocol have been proposed to overcome the difficult
problem of incomplete conversion. For example,
to reduce the likelihood of strand reannealing, the
genomic DNA may be imbedded in low melting
point agarose blocks (Olek et al. 1996); the bisulfite
reaction may be performed in a thermocycler with
repeated steps of heating to 95 ◦ C (Rein et al. 1997);
or a high concentration of urea may be added to
238

Figure 1. Principles of methylation analysis using MSREs (A) or bisulfite treatment (B). A: Genomic DNA is digested with a restriction
endonuclease that will not cut if the cytosine within the cleavage site is methylated. B: Treatment of genomic DNA with sodium bisulfite
deaminates unmethylated cytosine residues to uracil under conditions where 5-methylcytosine remains largely unaffected.

the bisulfite reaction (Paulin et al. 1998). Several
independent groups have confirmed the beneficial
effect of bisulfite treatment in agarose beads, whereas
the possible effects of the two latter modifications
remain controversial. Some genomic regions seem
to be particularly resistant to bisulfite conversion.
Warnecke et al. (2002) showed that such artifacts
might be less significant when DNA has been isolated
using a protocol involving extended proteinase K
treatment to remove residual protein.
Other potential artifacts of the sodium bisulfite
reaction should be considered when designing exper-
iments and interpreting the results. First, 2–5% of
5-methylcytosines may be converted to thymine in
the standard overnight incubation. This unintended
conversion is supposed to occur randomly and may
only become evident when individual DNA molecules
are analyzed. Second, treatment with bisulfite may
lead to DNA degradation due to partial acid-catalyzed
depurination. Long incubations can result in extensive
damage to template sequences, which subsequently
may be difficult to amplify (Raizis et al. 1995). Several
groups have reported that a 4-h incubation with
bisulfite may be sufficient for complete conversion,
provided that the DNA is completely denatured. Third,
when PCR is used to amplify bisulfite-treated DNA,
the same primer pair may not amplify the methylated
and unmethylated sequences with equal efficiency. At

present, there is no protocol that can be used to avoid
such biased PCR amplification, which should there-
fore be excluded experimentally by analyzing samples
containing both methylated and unmethylated DNA in
known ratios (Warnecke et al. 1997).
Techniques for gene-specific methylation analysis
A wide variety of methods have been developed to
assess the methylation status of specific genes in
a broad range of applications. While MSREs are
still widely used in combination with either Southern
analysis or PCR, most current methods rely on
treatment of genomic DNA with bisulfite prior to
PCR amplification. Several of these methods were
originally developed for detection of single-nucleotide
polymorphisms (SNPs) and disease-causing mutations
in genomic DNA, but later have been adapted for
detection of bisulfite-induced sequence differences
between methylated and unmethylated alleles.
MSRE-Southern analysis and MSRE-PCR
Analysis of gene-specific methylation based on initial
incubation of genomic DNA with MSREs may be
performed in two ways to determine the extent of
DNA digestion: Southern analysis and PCR. If large
amounts of DNA are available (∼10 µg), the digestion
products may be analyzed by conventional Southern
blotting. Essentially, the DNA digestion products
are fractionated by electrophoresis in an agarose gel,
blotted onto a nylon membrane and then hybrid-
ized with a radioactive probe representing the gene
of interest. If the site(s) of interest is methylated,
the bands generated with the methylation-sensitive
isoschizomer will differ in size from those generated
with the non-sensitive isoschizomer. The fraction of
digestion-resistant DNA may be quantified by image
analysis and is proportional to the degree of methyla-
tion present at that site (methylation level). The sensi-
tivity of Southern blotting analysis of MRSE-digested
DNA depends on the hybridization background, but is
usually ∼10%.
The sensitivity can be increased ∼1000-fold using
PCR. If the PCR primers are designed to flank the
restriction site, then a product will only be generated
if the site is not cleaved. To determine the fraction of
methylated molecules in the pool of DNA, the PCR
must be performed in a semi-quantitative format, for
example by including a homologues competitor in the
reaction, or by applying real-time quantitative PCR.
239
Several precautions must be taken to exclude false
positives resulting from an incomplete digest, in
particular when the digestion product is subjected to
PCR amplification. Because reduced accessibility of
DNA to cleavage is usually caused by impurities in the
DNA, the best way to ensure complete DNA cleavage
is careful purification of the DNA using proteinase
K. Although parallel cleavage reactions using the
two isoschizomers may serve as a good control for
complete cleavage, it cannot be excluded that the
different enzymes are affected differently by DNA
impurities. Even the inclusion of a control DNA frag-
ment with the same cleavage site as the target sequence
in the same reaction is not always sufficient to exclude
incomplete digestion. In general, because methylated
sites may not be distinguished from incomplete diges-
tion, MSRE methods should not be used for detection
of low-abundant methylated molecules.
Methylation-specific PCR
Methylation-specific PCR is currently the most
widely used technique to investigate the methylation
status of specific CpG sites in CpG islands (Herman
et al. 1996). The basic principle of this method
is PCR-based discrimination between methylated
and unmethylated DNA in bisulfite-converted DNA,
taking advantage of the bisulfite-induced sequence
differences (Figure 2). In a standard methylation-
specific PCR assay, two pairs of primers are included
in separate reactions to specifically amplify methyl-
ated and unmethylated molecules, respectively. Since
the two strands in bisulfite-treated DNA are no longer
complementary, primers should be designed specifi-
cally for one or both of these strands. After the PCR
has been completed, the products are usually analyzed
by agarose gel electrophoresis and ethidium bromide
staining.
The crucial step in methylation-specific PCR is
careful design of the primers; they must discrimi-
nate not only between methylated and unmethylated
molecules in bisulfite-treated DNA, but also between
bisulfite-converted DNA and DNA that has remained
unconverted during bisulfite treatment. To accom-
plish the former, one of the primers should contain a
CpG at its 3 end; to accomplish the latter, the other
primer should contain two non-CpG cytosines at its
3 end. Validation of individual methylation-specific
PCR assays should always include the appropriate
controls; methylated bisulfite-treated DNA, unmethyl-
ated bisulfite-treated DNA, and untreated genomic
240
Figure 2. Schematic outline of methylation-specific PCR. Genomic DNA is treated with bisulfite and used as template in a PCR containing
primers designed to discriminate between methylated and unmethylated alleles.
DNA. Genomic DNA universally methylated in vitro
using SssI DNA methyltransferase is a very useful
control for methylated alleles of any gene.
As long as the bisulfite protocol used for conver-
sion of DNA does not lead to extensive DNA degrada-
tion due to depurination, methylation-specific PCR
requires only small quantities of DNA as starting
template. Furthermore, methylated molecules can be
detected down to a level of 0.1% in the total popula-
tion. By simultaneous amplification of unmethyl-
ated and methylated molecules in the same reaction
tube, it may be possible to make a semiquantitative
assessment of allele types. Nevertheless, the infor-
mation obtained by methylation-specific PCR should,
in general, be considered only qualitative, and the
results should be further validated using a quantitative
or semiquantitative method (see below). Methylation-
specific PCR is, therefore, best suited as a rapid and
cost-effective method to initially screen samples for
methylation of specific genes. Another limitation is
that usually only a single CpG site is reliably probed,
and that CpGs outside CpG islands are not amenable
to analysis.
Methylation-sensitive single-nucleotide primer
extension (Ms-SnuPE)
Ms-SnuPE (Gonzalgo and Jones 1997) is a tech-
nique that allows direct quantification of the degree of
methylation at virtually any CpG site by combining
bisulfite treatment of DNA with the classical SnuPE
assay (Kuppuswamy et al. 1991). The latter technique
was developed for detection of single-base mutations
and utilizes an oligonucleotide primer that anneals
to a PCR-generated template immediately 5 to the
nucleotide of interest. The annealed primer is then
extended in the presence of DNA polymerase and
an appropriately labeled deoxyribonucleotide. The
relative amount of incorporated label is proportional
to the relative amount of that particular base at that
particular site in the original template.
In the classical Ms-SnuPE protocol (Figure 3),
genomic DNA is treated with sodium bisulfite
followed by strand-specific PCR to yield a suitable
DNA template of the target sequence. Amplification
must be performed using PCR primers that are specific
for bisulfite-converted DNA and do not discriminate
between methylated and unmethylated alleles. The
former is accomplished by including many non-CpG
cytosines (converted into uracil by bisulfite) in the
primer binding sites; the latter is usually accomplished
by avoiding CpG sites in the primer binding sites. The

resulting PCR product is purified following separa-
tion in agarose gels and is then hybridized with a
primer flanking the CpG site(s) being investigated.
To prevent unwanted bias, oligonucleotides used in
the primer extension reactions should not anneal to
sequences that originally contained one or more CpG
sites, which may not always be possible in CpG-
dense regions. After the primer has hybridized to its
target sequence, a single-nucleotide extension reac-
tion is performed in the presence of Taq polymerase
and either [α-32 P]dCTP or [α-32 P]dTTP. Quantifica-
tion of the ratio of methylated versus unmethylated
cytosine (C vs. T) at the original CpG sites can be
determined by denaturing polyacrylamide gel electro-
phoresis and phosphorimager analysis. Alternatively,
Ms-SnuPE reaction products can be transferred to
nylon membranes for quantification.
Although fraught with the technical problems of
the bisulfite method (incomplete conversion, PCR
bias, etc.) and some experimental pitfalls (Oakeley
1999), Ms-SnuPE has several advantages over other
methods for determining methylation levels: it is
quantitative; it avoids the use of restriction enzymes;
and small amounts of DNA can be analyzed. Further-
more, the methylation status of several CpG sites
can be assessed simultaneously by including multiple
oligonucleotides in a single primer extension reaction.
El Maarri et al. (2002) described a non-radioactive
variant of Ms-SnuPE, in which ion-pair reverse phase
HPLC is used to discriminate and quantify the
extended primer products on the basis of different
masses and hydrophobicities. After annealing to
bisulfite-treated DNA, the primers are extended in
the presence of both ddCTP and ddTTP. In order
to quantify the incorporation of either ddTTP or
ddCTP, the extension products are loaded directly
onto a denaturing high performance liquid chroma-
tography (DHPLC) column that allows separation of
non-extended, ddCTP-extended, and ddTTP-extended
primers.
Bisulfite genomic sequencing
Sequence analysis of PCR products generated from
bisulfite treated DNA (Frommer et al. 1992) is consid-
ered the gold standard for gene-specific methylation
analysis, first of all because it provides information
about the methylation status of every cytosine residue
within the target sequence. For highest resolution of
methylation patterns, the PCR products are cloned into
an appropriate plasmid vector followed by sequence
241
analysis of a number of individual clones. While
the experimental procedure is laborious and time-
consuming, interpretation of data is simple: the pres-
ence of a band in the C-track indicates the presence of
a methylated cytosine in the corresponding genomic
DNA (Figure 4A). Alternatively, the PCR product may
be sequenced directly to provide an average across
all molecules in the sample. The presence of bands
in both the C- and T-tracks at a specific position in
the sequence indicates partial methylation. However,
as pointed out by Oakeley (1999), care should be
taken when using the relative intensities of these
bands to estimate the proportion of 5-methylcytosine
to cytosine in the genome. The major disadvantages
of cloning and sequencing are that a high number
of clones have to be sequenced for reliable results,
and that artifacts relating to PCR infidelity, incom-
plete bisulfite conversion, or bisulfite conversion of 5-
methylcytosine to thymine become significant. Direct
sequence analysis is less prone to artifacts but does not
provide information about the methylation patterns of
individual alleles.
MethyLight
MethyLight (Eads et al. 2000) is a high-throughput
DNA methylation analysis technique that relies on
fluorescence-based real-time PCR. After bisulfite
conversion, the DNA is amplified by PCR in a reaction
containing three oligonucleotides; a probe flanked by
a forward primer and a reverse primer, all of which
are specific for the target. The probe is dual-labeled
with a 5 fluorescent reporter dye and a 3 quencher
dye. When the probe is intact, the quencher dye
absorbs the fluorescence of the reporter dye due to
their proximity. After annealing to the PCR product,
the probe is cleaved by the 5 to 3 exonuclease
activity of Taq DNA polymerase. This cleavage will
release the reporter from the quencher, resulting in an
increased fluorescence signal, which is proportional to
the amount of PCR product generated. The fluores-
cent signal is measured at every cycle, and the initial
template methylation level can be derived from the
cycle number at which the fluorescent signal crosses
the detection threshold in the exponential phase of the
PCR reaction.
The MethyLight technique has several important
advantages. First, it is a highly sensitive assay, capable
of detecting methylated alleles in the presence of a
10,000-fold excess of unmethylated alleles, which
allows identification of aberrant DNA methylation
242

Figure 3. Schematic outline of Ms-SnuPE. Genomic DNA is treated with bisulfite followed by PCR amplification using primers that do
not discriminate between methylated and unmethylated alleles. The PCR product is then incubated with a primer that anneals to sequences
upstream and terminates immediately 5 to the site being monitored. After primer annealing, a single-nucleotide extension reaction is performed
in the presence of Taq polymerase and [α-32 P]dTTP or [α-32 P]dCTP. The radiolabeled reaction products are typically electrophoresed in
polyacrylamide gels and quantified by phosphorimage analysis.

patterns in samples contaminated with substantial Methylation-specific single-strand conformation

amounts of normal DNA. Second, the quantitative
format allows accurate information about the fraction
of DNA molecules harboring a particular methyla-
tion pattern in the total pool of molecules. Third, the
risk of PCR contamination is significantly reduced
because no manual transfer of PCR products is
required. Fourth, sequence discrimination may be
achieved at multiple levels, depending on the choice
of primers and probes. In addition to the need for an
expensive instrument, the drawbacks of this method
are that it requires expensive hybridization probes,
that serial dilutions of fully methylated and fully
unmethylated control samples must be included in
each experiment to generate standard curves, and
that heterogeneous DNA methylation may not be reli-
ably detected. Accordingly, the MethyLight technique
is not designed to yield high-resolution methylation
information; rather, its strongest features are its high-
throughput capabilities and its high sensitivity.
analysis (MS-SSCA)
Single-strand conformation analysis (SSCA), also
known as single-strand conformation polymorphism
(SSCP), is one of the most widely used techniques for
detection of mutations in genomic DNA. Its popularity
is primarily due to the simple and rapid procedure
that requires no special equipment. The basic
protocol involves denaturation of the PCR product
and subsequent electrophoresis in a non-denaturing
polyacrylamide gel. The migration distance of a
single-stranded DNA molecule is determined by its
secondary structure, which is highly dependent on the
primary nucleotide sequence. Even single-base substi-
tutions may significantly alter the secondary structure
of single-stranded DNA and hence cause a shift in
electrophoretic mobility (Orita et al. 1989).
In MS-SSCA (Bianco et al. 1999; Maekawa et
al. 1999; Burri and Chaubert 1999), genomic DNA

Figure 4. Methods to resolve sequence differences in PCR products generated from bisulfite-treated DNA using primers that do not discriminate
between methylated (M) and unmethylated (U) alleles.
is treated with sodium bisulfite, and the region of
interest is amplified using primers that do not discrimi-
nate between methylated and unmethylated alleles.
The bisulfite-induced sequence differences between
methylated and unmethylated alleles can then be
resolved by denaturation of the PCR product into
single strands followed by electrophoresis in a poly-
acrylamide gel under non-denaturing conditions
(Figure 4B). After electrophoresis, bands can be visu-
243
alized using high-sensitivity fluorescent gel stains, e.g.
SYBR Green II and SYBR Gold. Ethidium bromide
binds poorly to single-stranded DNA and is usually
not recommended for staining SSCP gels, in particular
when denaturation of the PCR product is incomplete
and/or re-annealing of the strands is extensive. Effi-
cient denaturation and prevention of re-annealing both
may be achieved by denaturing the PCR product in
a loading buffer containing 95% formamide and 10
244
mM sodium hydroxide at 95 ◦ C for 5 min, and then
immediately placing the sample on ice.
One of the great advantages of SSCA is that
alleles with different methylation patterns are physi-
cally separated in the gel and may be extracted
for subsequent sequence analysis. This provides a
simple means for identifying individual epigeno-
types in a pool of DNA without time-consuming
cloning analysis. The limitation of MS-SSCA is that
probably not all methylation changes are resolved.
First, conventional SSCA analysis has a specificity
of <70%. Second, in unmethylated bisulfite-treated
DNA, the number of bases is reduced from 4 to 3,
which significantly reduces the ability of the single
stranded molecules to form secondary structures.
Conversion of non-CpG cytosines may also reduce
or entirely abolish internal base pairing of methyl-
ated sequences. Until more systematic studies have
clarified this issue, MS-SSCA may not be considered
a generally applicable method for resolving gene-
specific methylation changes.
Combined bisulfite restriction analysis (COBRA)
COBRA (Xiong and Laird 1997) is a powerful tech-
nique that exploits differences in restriction enzyme
recognition sites between methylated and unmethyl-
ated DNA after treatment with bisulfite (Figure 4C).
Bisulfite conversion can lead to both methylation-
dependent creation of new restriction enzyme sites and
methylation-dependent retention of pre-existing sites.
After bisulfite treatment, the sequence of interest is
amplified by PCR using primers that do not discrimi-
nate between methylated and unmethylated alleles.
The resulting PCR products will represent a mixed
population of methylated and unmethylated alleles.
The fraction of molecules that has a newly created
or retained restriction site containing a CpG directly
reflects the methylation level at that site in the original
pool of genomic DNA. The PCR products are then
digested with a restriction enzyme that has cytosines
only within CpG sites in its recognition sequence. For
example, TaqI cleaves the sequence TCGA, which will
be retained in bisulfite-treated DNA if the cytosine is
methylated, but will be transformed to TTGA if the
cytosine is unmethylated. By appropriate choice of
restriction site and enzyme, a positive result (cleavage)
can be varied to indicate either non-methylation or
methylation.
The digestion products may be analyzed by
agarose gel electrophoresis and ethidium bromide
staining. For semi-quantitative analysis, the digested
PCR products may be denatured, separated by
denaturing polyacrylamide gel electrophoresis, and
then transferred to a membrane by electroblotting
and hybridized with radioactively labeled oligonucle-
otides. Quantification can be performed using a
phosphorimager, and the percentage of fully methyl-
ated sites in a genomic DNA sample can be calculated
as the ratio between the cleaved PCR product and the
total amount of PCR product. For optimal restriction
enzyme digestion, purification of the PCR products
may be necessary.
COBRA analysis allows the determination of DNA
methylation levels at specific loci in small amounts
of genomic DNA, provided that the DNA is not too
extensively degraded during bisulfite treatment. Even
though COBRA is based on the use of restriction
enzymes, it is not nearly as prone to false positives
as MSRE-PCR because it does not rely on the absence
of cleavage to detect methylated sites. As with most
other bisulfite methods, incomplete bisulfite conver-
sion of the DNA and PCR bias will severely affect
the reliability of the COBRA technique. An additional
limitation is that it is limited to restriction sites and
thus cannot be used to analyze the methylation status
of all possible CpG sites.
Methylation-specific denaturing gradient gel
electrophoresis (MS-DGGE)
Denaturing gradient gel electrophoresis (DGGE) is
widely used in combination with PCR as a tech-
nique to detect single-base mutations in genomic
DNA (Abrams and Stanton 1992). Separation of DNA
molecules by DGGE is based on the electrophoretic
mobility of a partially denatured (melted) molecule in
polyacrylamide gels, which is decreased significantly
compared with that of the corresponding helical form.
In a gel containing an increasing gradient of chem-
ical denaturants, the migration of the molecules will
almost stop at the position at which the transition from
the helical to the partially melted form occurs. Single-
base substitutions and other sequence alterations can
significantly alter the melting temperature, causing
the variant molecules to stop migrating at different
positions in the denaturing gradient gel.
MS-DGGE (Aggerholm et al. 1999) resolves dif-
ferentially methylated DNA molecules on the basis of
differences in melting temperature after bisulfite treat-
ment (Figure 4D). After non-discriminatory amplific-
ation of methylated and unmethylated sequences, the

PCR product contains a mixture of molecules with
different contents of G:C base pairs, reflecting the
distribution of methylated CpGs in the genomic DNA
template. The PCR products are then resolved by
electrophoresis in a polyacrylamide gel containing a
gradient of urea and formamide, which is kept at a
constant temperature of 54–60 ◦ C. Because a G:C base
pair contains three hydrogen bonds and is more stable
than an A:T base pair with only two hydrogen bonds,
the migration distance of the PCR product increases
with an increase in methylation density. After electro-
phoresis, the gel is stained with ethidium bromide or a
high-resolution fluorescent gel stain.
An attractive feature of MS-DGGE is that
sequences with different degrees of methylation will
be retarded at different positions in the gel. This
provides a detailed visual display of the methylation
status of the total pool of DNA molecules. This
is particularly useful for resolving heterogeneous
methylation (Aggerholm et al. 1999). Furthermore,
like with MS-SSCA, different epigenetic clonotypes
will be revealed as distinct bands in the gel, which may
be recovered for subsequent sequence analysis. The
drawback of this method is the need for careful design
of PCR primers to ensure optimal melting behavior of
the amplified product. In general, it is recommended
that the primers be designed to generate a PCR
product that, for both unmethylated and methylated
sequences, contains two distinct melting domains:
a high-melting domain and a low-melting domain
containing the entire sequence of interest. Introduc-
ing a new high-melting domain and converting the
sequence of interest into a single low-melting domain
may both be accomplished by PCR-mediated attach-
ment of a GC-rich sequence, generally known as
a GC-clamp (Sheffield et al. 1989). The impact of
adding a GC-clamp to one of the ends of a PCR
product can be accurately predicted by using the
computer algorithm MELT87 (Lerman and Silverstein
1987).
Methylation-specific melting curve analysis
(MS-MCA)
MS-MCA (Worm et al. 2001) is an in-tube assay
that, like MS-DGGE, exploits differences in melting
temperature between methylated and unmethylated
alleles after bisulfite treatment. Essentially, non-
discriminatory amplification of the bisulfite-treated
template is performed in the presence of a PCR-
compatible, fluorescent dye that specifically binds
245
to double-stranded DNA, e.g. SYBR Green I. The
melting properties of the PCR product can be
examined immediately after amplification by slowly
elevating the temperature under continuous fluores-
cence monitoring. An abrupt decrease in fluorescence
reflects the cooperative melting of a single domain in
the PCR product and can be identified if the melting
curves are converted into melting peaks by plotting the
negative derivative of fluorescence over temperature
versus temperature. The 5-methylcytosine content of
the genomic DNA template determines the absolute
position of the PCR product’s melting peak (Figure
4E).
The major advantages of MS-MCA are that it
requires only standard, inexpensive PCR reagents; it
resolves heterogeneous methylation both in pools of
DNA and in cloned PCR products; and it detects
methylated and unmethylated alleles in the same
reaction. PCR amplification and subsequent melting
analysis may even be integrated using a thermal
cycler coupled with a fluorometer (LightCycler or
equivalent). The major advantage of this integrated
procedure, compared with existing gel-based methods,
is the avoidance of post-PCR experimental steps,
which also reduces the risk of PCR contamination.
The major limitation of this method is that no informa-
tion is provided on the methylation status of individual
cytosines, and that the sensitivity is relatively low,
making it unsuitable for detection of low-abundant
methylated alleles. In addition, like in MS-DGGE,
manipulation of the PCR product’s melting properties
may be required to ensure that the entire region of
interest melts as a single peak.
Methylation-specific denaturing high-performance
liquid chromatography (MS-DHPLC)
DHPLC (Xiao and Oefner 2001) was developed as a
mutation-detection technique and has later been
adapted for detection of changes in DNA methyla-
tion patterns. In the original protocol for analysis of
genomic DNA, mutant and wild-type amplicons are
subjected to analysis on reversed-phase chromatog-
raphy supports under partially denaturing conditions
and detected by on-line UV or fluorescence monitor-
ing. The stationary phase, which has become commer-
cially available, is made of alkylated nonporous
poly(styrene-divinylbenzene) particles. Separation of
DNA molecules is achieved by means of a hydro-
organic eluent containing triethylammonium and
acetate ions. At elevated temperatures, retention of
246
DNA molecules is sequence dependent, forming the
basis for resolution of sequence differences (Xiao and
Oefner 2001).
Deng et al. (2000) and Baumer et al. (2001)
showed that DHPLC is also useful for resolution of
differences in DNA methylation. After bisulfite treat-
ment of DNA, the region of interest is amplified using
non-discriminatory primers, and the amplicon is then
applied directly onto the DHPLC column. Ampli-
cons generated from methylated sequences have a
higher G:C-content and, therefore, melt less and elute
later than the corresponding unmethylated sequences.
DHPLC is a rapid and cost-effective technique for
high-throughput analysis of DNA methylation. The
disadvantages include the need for expensive, special-
ized equipment and the use of the toxic substance,
acetonitrile. At present, little is known about the
sensitivity of MS-DHPLC.
Methylation-specific microarray (MSO)
MSO uses bisulfite-treated DNA as template for
non-discriminatory PCR amplification, followed by
hybridization of the PCR product to glass slides
carrying oligonucleotides that discriminate between
methylated and unmethylated cytosines at specific
CpG positions (Gitan et al. 2002; Adorjan et al.
2002). To allow detection of hybridization signals
by fluorescence analysis, the amplification primers
carry a fluorescent label. Each of the detection oligo-
nucleotides is designed to hybridize to the bisulfite-
modified sequence around one CpG site, which is
either originally unmethylated (TG) or methylated
(CG). Hybridization conditions must be selected to
allow detection of the single nucleotide differences
between the TG and CG oligonucleotides. One of the
great potentials of MSO is that multiple genes can be
analyzed on the same array. For example, Adorjan et
al. (2002) assessed the methylation status of 232 indi-
vidual CpG sites representing 56 different genes as a
means to distinguish different solid tumors. A poten-
tial limitation of this method is that closely spaced
CpGs may not be amenable to analysis if the gene in
question is heterogeneously methylated.
Global CpG island methylation analysis
Previous attempts to identify new genes that are differ-
entially methylated in human disease have primarily
been candidate gene approaches relying on the use
of techniques for gene-specific methylation analysis.
Within recent years, however, new high-throughput
methods have made it possible to simultaneously
analyze the methylation status of thousands of CpG
islands in a pool of DNA.
Restriction landmark genomic scanning (RLGS)
RLGS is a method that provides a bird’s-eye view of
the methylation status of thousands of unselected CpG
islands in the genome within a single gel (Costello
et al. 2000). Genomic DNA is initially digested
with methylation-sensitive restriction enzymes, such
as NotI, which recognize large CpG-rich sequences.
These sequences usually occur in CpG islands and are
cleaved only if the CpG dinucleotides are unmethyl-
ated. The digested DNA is radioactively labeled at
the restriction sites using [α-32 P]dCTP, [α-32 P]dGTP
and DNA polymerase, digested with a second restric-
tion enzyme, such as EcoRV, and then subjected to
electrophoresis in an agarose tube gel (first-dimension
separation). The agarose gel is then equilibrated in
HinfI digestion buffer and the DNA digested in the gel
with HinfI. The agarose gel is then placed horizon-
tally across the top of a nondenaturing polyacrylamide
gel, and the DNA is separated by electrophoresis in
the second dimension. The gels are then dried and
exposed to X-ray film or inspected using a phosphor-
imager.
The above approach produces a complex pattern
of spots, in which a spot will be missing if a particular
NotI site is methylated and has not, therefore, been
recognized and cleaved. This spot may be stabbed
from the corresponding control gel for further identifi-
cation by sequence analysis. Alternatively, software
systems have been developed for automated analysis
of RLGS profiles. Differences between RLGS profiles
have been used to identify imprinted genes and genes
involved in disease states such as cancer.
One of the most critical parameters in RLGS is
DNA quality; even small amounts of degraded DNA
may lead to a significant reduction in the quality of
the RLGS profiles. Other disadvantages include the
labor-intensive and time-consuming procedure, the
need for large amounts of starting DNA, and the diffi-
cult interpretation of the two-dimensional gel images.
Furthermore, although the technique is useful for
simultaneously assessing the methylation status of a
large number of CpG sites, it does not provide infor-
mation about whether or not a given CpG island
resides within the promoter region of a gene. Accord-

ingly, only a proportion of the aberrantly methylated
CpG islands are associated with transcriptional gene
silencing.
Methylated CpG island
amplification-representational difference analysis
(MCA-RDA)
MCA (Toyota et al. 1999) is a PCR-based method
that allows preferential amplification of closely spaced
methylated SmaI sites, which occur predominantly in
CpG islands. Unmethylated SmaI sites in genomic
DNA are first eliminated by digestion with SmaI,
which cuts only when CpG sites within the target
sequence are not methylated, and leaves the fragment
blunt ended. DNA is then digested with the SmaI
isoschizomer, XmaI, which digests at methylated sites
and leaves an overhang. Adaptors are then ligated to
these overhangs, and PCR is performed using primers
complementary to these adaptors. The MCA ampli-
cons represent a pool of methylated CpG islands that
can be used directly in a dot blot analysis to study the
methylation status of any gene for which a probe is
available. Alternatively, to identify CpG islands whose
methylation status differs between normal DNA and
DNA from pathological tissues, MCA can be coupled
with representational difference analysis (RDA), a
subtractive hybridization technique designed to isolate
genomic differences between two complex genomes
(Lisitsyn et al. 1993). The subtractive and kinetic
enrichment of differentially methylated sequence by
RDA has the potential to identify sequences methyl-
ated only in pathological conditions.
Like RLGS, the MCA-RDA method can be useful
for simultaneously determining the methylation status
of multiple CpG-dense loci. It should be noted,
however, that only those SmaI sites that are <1 kb
apart can be amplified using MCA. On the one hand,
this will ensure that the most CpG-rich sequences are
represented in the MCA amplicons; on the other hand,
∼25% of all possible CpG islands do not contain
two closely spaced SmaI sites. Other disadvantages
of MCA-RDA are that it examines only a limited
number of CpG sites within a given CpG island, and
that ∼60% of the recovered clones are Alu repetitive
sequences.
Methylation-sensitive arbitrarily primed PCR
(MS-AP-PCR)
MS-AP-PCR (Gonzalgo et al. 1997) is a PCR-based
fingerprinting method that can be used to screen
247
for methylation changes and to isolate specific DNA
fragments associated with these changes. This method
relies on digesting genomic DNA with methylation-
sensitive and insensitive restriction enzymes followed
by amplification with random primers that preferen-
tially bind to sequences associated with CpG islands.
A similar approach was described by Huang et al.
(1997), who called it methylation-sensitive restriction
fingerprinting (MSRF). According to the protocol of
Gonzalgo et al. (1997), genomic DNA is digested
in three independent reactions, containing RsaI,
RsaI + HpaII, and RsaI + MspI, respectively, and
subsequently amplified and radioactively labeled with
GC-rich primers under low-stringency PCR conditions
in the presence of [α-33 P]dATP. The resulting PCR
products are resolved on high-resolution polyacryla-
mide gels under denaturing conditions, followed by
exposure of the dried gel to autoradiographic film.
Fragments that show differential methylation can be
stabbed from the gel and identified by sequence
analysis. Separate digestions of DNA using the
methylation-sensitive enzyme, HpaII, and its methyla-
tion-insensitive isoschizomer, MspI, are important for
controlling for the presence of PCR products unrelated
to methylation changes. Double digestion with either
of these enzymes in combination with RsaI is used to
generate smaller fragments. MS-AP-PCR can detect
methylation changes in as little as 200 ng of genomic
DNA, but like RLGS and MCA-RDA it does not
provide information about the possible associations
between methylation changes and changes in gene
expression.
Differential methylation hybridization (DMH)
Future studies on global changes in DNA methylation
will probably be speeded up by the use of microarray-
based technologies. The principle of one such method,
DMH, was first described by Huang et al. (1999)
and has since been further developed for large-scale,
global analysis using a microarray format (Yan et al.
2001). Genomic DNA is pre-cut with a methylation-
insensitive enzyme, such as MseI. Linkers are then
ligated to the digested DNA before it is incubated with
the methylation-sensitive enzymes, BstUI and HpaII.
The resulting digests are amplified by PCR and the
products hybridized to an array of immobilized CpG
islands. Yan et al. (2001) used a microarray containing
∼8,000 GC-rich tags to identify epigenetic alterations
in breast cancer.
248
So many techniques – how to choose?
Mapping of 5-methylcytosine in genomic DNA has
become an important tool for studying the molecular
basis of human disease and for understanding tissue-
specific gene expression. However, to generate reli-
able data on DNA methylation is by no means a trivial
task. First, the cellular heterogeneity of most tissues
makes it difficult to assign a particular methylation
signal to a specific cell type. Second – as revealed
by the plethora of available techniques – no single
technique fulfills all criteria for generating unambigu-
ous data on DNA methylation. The purpose of this
review has been to provide an overview of current
key techniques and describe the most important advan-
tages and disadvantages, which should be carefully
considered before deciding on which approach would
best provide a useful set of data. Some of these
techniques are technically challenging, and choice
of methodology will often depend on the available
equipment and expertise.
The quality and quantity of the starting DNA
are important limiting factors in DNA methylation
analyses; several of the techniques described in this
review require >1 µg of high-quality DNA from
cultured cells or fresh tissue samples for reliable
results. Accordingly, due to the limited quantity and
extensive degradation of DNA, only sparse infor-
mation on DNA methylation may be derived from
archived pathology samples, such as formalin-fixed,
paraffin-embedded tissue sections. Degradation of
DNA during bisulfite treatment may further reduce
the number of intact DNA molecules amenable to
PCR analysis, and two rounds of amplification using
nested or semi-nested primers are usually necessary to
generate sufficient amounts of the product. The low
number of starting molecules may result in stochastic
amplification, leading to the generation of a PCR
product that does not accurately reflect the distribu-
tion of methylated and unmethylated molecules in the
original sample. Millar et al. (2002) have described a
number of bisulfite protocols that may help optimizing
the analysis of archived samples. Nevertheless, data
on DNA methylation obtained from limited material
must always be interpreted with great caution, unless
the results can be verified in several independent
experiments.
For gene-specific methylation analysis, where the
target under investigation usually is an aberrantly
methylated CpG island, it is important to consider how
detailed the resolution of 5-methylcytosine must be
to provide the necessary information. For example,
neither extensive analysis of the methylation pattern
nor sophisticated quantitative analysis are required to
make a diagnosis of an imprinting disorder, in which
either methylated or unmethylated alleles are entirely
missing in all cells. In this case, it may be fully
sufficient to rely on a technique with relatively low
sensitivity, such as MSRE-Southern analysis. On the
other hand, highly sensitive techniques are required
for detection of low-abundant, aberrantly methylated
alleles in aging or cancer tissues, or in DNA released
from neoplastic and preneoplastic lesions into serum,
urine or sputum. Usually, the most rational approach is
to use methylation-specific PCR to determine whether
or not aberrantly methylated alleles are present in
the sample. Once a positive signal has been obtained
using methylation-specific PCR, further quantitative
or semi-quantitative analysis is necessary to exclude
false positives. In-depth resolution of methylation
patterns at the nucleotide level requires sequence
analysis of PCR products.
Unfortunately, bisulfite-based procedures for
detection of aberrant DNA methylation are neither
trivial nor foolproof. As described in detail above, a
number of potential artifacts and pitfalls should be
carefully considered, including PCR bias, incomplete
conversion of cytosine, and unintended conversion
of 5-methylcytosine. Furthermore, PCR assays based
on bisulfite treatment of DNA may not be easily
established. Li and Dahiya (2002) have developed
a program, MethPrimer, that may be useful for
designing primers to specifically amplify bisulfite-
treated DNA. In its present version, this program
is useful for designing primers for methylation-
specific PCR, but further development is undergoing
to make it supportive for COBRA and Ms-SnuPE as
well. MethPrimer has been made freely accessible
(http://itsa.ucsf.edu/˜urolab/methprimer/).
A particularly powerful tool for identifying an
aberrantly methylated cell type in a complex tissue
is bisulfite analysis of microdissected cells. Indeed,
bisulfite treatment of DNA in agarose beads has
been used to generate detailed methylation patterns
of single cells representing different stages of male
germ cell differentiation (Kerjean et al. 2000). The
combination of microdissection and bisulfite analysis
is likely to significantly increase our knowledge about
the role of DNA methylation in development, disease
and aging.

Acknowledgements
We thank J. Worm and K. Grønbæk for critical
comments on this manuscript. Supported by the
Danish Cancer Society, the Danish Medical Research
Council, the Kaarsen Foundation, and the Novo
Nordisk Foundation.
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