Review

O
N

Optimizing methodologies for PCR-based DNA

methylation analysis

LY

Review

IO
N

Hernn G. Hernndez1,2,3, M. Yat Tse4, Stephen C. Pang4, Humberto Arboleda2, and Diego A. Forero1
1
Laboratory of NeuroPsychiatric Genetics, Biomedical Sciences Research Group, School of
Medicine, Universidad Antonio Nario, Bogot, Colombia, 2Neurosciences Research Group,
School of Medicine, Universidad Nacional de Colombia, Bogot, Colombia, 3Biomedical Sciences
Doctoral Program, School of Medicine, Universidad Nacional de Colombia, Bogot, Colombia,
4
Department of Biomedical and Molecular Sciences, Queens University, Kingston, ON, Canada.

BioTechniques 55:181-197 (October 2013) doi 10.2144/000114087

Keywords: epigenomics; DNA methylation; MS-HRM; MethyLight; 5-methylcytosine; polymerase chain reaction; reference standards

melting (MS-HRM). These techniques do

not need expensive specialized equipment
and could be implemented in a typical
molecular genetics laboratory.

R
E

R
IN

IT

DNA methylation is the most extensively

studied mechanism for epigenetic gene
regulation (1). Recent studies have shown
that DNA methylation plays an important
role in a number of physiological processes
as well as common diseases such as cancer
and neurodegenerative disorders (2,3). In
mammals, DNA methylation occurs at the
C-5 position of cytosine in CpG dinucleotide sequences (Figure 1) (1), which are
mainly concentrated in regions known as
CpG islands. Methylation in CpG islands
within gene promoters usually leads to gene
silencing. More recently, DNA methylation
in regions located up to 2 kb from known
CpG islands (called CpG island shores) has
also shown a strong correlation with gene
expression (4).
At present, the vast array of platforms
available to study DNA methylation present
a challenge for scientists who wish to enter
this field (5). Among the methods for
studying DNA methylation in candidate
regions, PCR-based approaches have
several advantages (6). Here we provide
a practical overview of experimental
design and analysis for the most common
PCR-based DNA methylation techniques:
bisulfite sequencing PCR (BSP), methylation specific PCR (MSP), MethyLight,
and methylation-sensitive high resolution

IS

Comprehensive analysis of DNA methylation patterns is critical for understanding the molecular basis of many human diseases. While hundreds of PCR-based DNA methylation studies are published every year, the selection and
implementation of appropriate methods for these studies can be challenging for molecular genetics researchers not
yet familiar with methylation analysis. Here we review the most commonly used PCR-based DNA methylation
analysis techniques: bisulfite sequencing PCR (BSP), methylation specific PCR (MSP), MethyLight, and methylation-sensitive high resolution melting (MS-HRM). We provide critical analysis of the strengths and weaknesses
of each approach as well as a series of guidelines to assist in selecting and implementing an appropriate method.

Vol. 55 | No. 4 | 2013

Bisulfite conversion

The first step in almost all protocols for

studying DNA methylation is bisulfite
conversion of the DNA sequence of interest.
Bisulfite conversion occurs through a
number of chemical reactions (e.g., sulfonation, deamination, and desulfonation) on
the DNA that transform non-methylated
cytosines into uracils. Methylated cytosines
remain unconverted (Figure 1). Classical
DNA conversion protocols are timeconsuming, often requiring more than
16 h to complete (7), and require multiple
tube changing steps that increase the risk of
contamination and human error. Classical
protocols also risk losing more than 75%
of the starting DNA (8,9) during purification and through single-strand breaks
that occur during long incubation steps
(7,9).
Commercially available bisulfite
conversion kits improve recovery of the
converted DNA by using shorter incubation
steps and alternative purification procedures (9). These kits also facilitate efficient

181

implementation of the conversion reaction,

thereby improving downstream results
with PCR-based techniques. Thus, kits
are highly recommended, especially for
those unfamiliar with this field of study.
There are many considerations for selecting
a kit, including cost, yield, efficiency, and
time. A comparison of the main features of
available DNA conversion and methylation
control kits is included in Tables 1 and 2.
Controls for DNA bisulfite conversion
Evaluation of the quality of converted
DNA is recommended when beginning
a DNA methylation study; this step is
especially important for quantitative
PCR-based methods such as MethyLight
and MS-HRM. Since bisulfite-treatment
can result in DNA fragmentation, thus
reducing the number of molecules available
for PCR amplification, it is best to test the
bisulfite-converted DNA with primer sets
that amplify a range of differently sized
products. From these products, the ideal
amplicon length for downstream analysis
can be determined (10), providing information that will aid in primer design.
Incomplete bisulfite conversion
will adversely affect the reliability and
accuracy of DNA methylation measurements by PCR-based methods (11,12).
www.BioTechniques.com

Figure 1.
Bisulfite conversion

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Non-converted sequence

Methylated DNA
CGNNCNNNCNNCGCNCG

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NA_Meth_one_third_vert_Biotechniques.indd 1

Post bisulfite treatment

sequences

CH3

CH3

CH3

Non Methylated DNA

CGNNCNNNCNNCGNNCG

UGNNUNNNUNNUGUNUG

Figure 1. Outline of bisulfite conversion. Non-methylated cytosines are transformed to thymines.

N represents a nucleotide unchanged by bisulfite treatment. A light blue U represents a uracil
derived from bisulfite conversion of non CpG cytosines. A red U represents a uracil derived from
bisulfite conversion of non-methylated cytosines in a CpG dinucleotide. Methylated cytosines in a
CpG dinucleotide are not modified by the bisulfite conversion reaction. The CpG dinucleotides and
the UpG dinucleotides derived from the bisulfite conversion reaction are in bold and underlined.

Therefore, it is necessary to evaluate the

efficiency of conversion using commercially available primer sets to amplify the
converted DNA (e.g., DAPK1 Catalog
#D50142, Zymo Research, Orange, CA)
(Table 1). The resulting DNA product
can be sequenced to verify the efficiency
of conversion for all non-CpG cytosines.
Alternatively, converted DNA may be
amplified with primers designed for the
non-converted DNA sequence. In this case,
the absence of a PCR amplicon suggests a
complete conversion reaction.
Converted DNA must also be quantified
prior to downstream PCR applications. The
amount of DNA may be determined by
spectrophotometric measurements using
the NanoDrop 2000 spectrophotometer
(Thermo Scientific, Waltham, MA) (13)
with settings for single-stranded DNA,
or agarose gel electrophoresis and classical
UV spectrometric analyses (5). Other more
specific methods, such as qPCR (including
MethyLight control assays) or PicoGreen
may be more reliable and better suited for
measuring limited amounts of DNA (14).
Designing primers for PCR-based
DNA methylation analysis
Designing primers against a region of
interest (ROI) is the most critical step in
obtaining adequate DNA methylation
results using PCR-based methods. Several
software platforms such as Methyl Primer
Express (Applied Biosystems, Foster City,
CA), MethPrimer (15), BiSearch (16),
MethMaker (17), and MSPprimer (18)
have been developed for this purpose. All
of these programs allow users to customize
primer length, amplicon length, and Tm
(melting temperature) differences, as well
as enable searches for CpG islands in the
input sequence, and identify possible
stable primer-dimer or hairpin structures
that should be avoided. The advantages
and disadvantages of each program are

182
9/16/13 12:49 PM

CGNNUNNNUNNCGUNCG

compared in Table 3. Primers should not

bind to regions containing common SNPs
(19), which can be identified easily using the
UCSC Genome Browser (http://genome.
ucsc.edu).
Because bisulfite treatment decreases
DNA sequence complexity, primers have
an increased tendency to bind multiple
target sequences in converted DNA (18).
Therefore, in silico evaluation of primer
specificity is a key step during primer design
for bisulfite-converted DNA methods.
BiSearch software is unique in terms of
its ability to find the number of potential
matches, including partial matches, for
each individual primer in the bisulfiteconverted methylated or unmethylated
genome and to perform in silico PCR on the
bisulfite-converted human genome using
any primer pair (16). In our laboratory, we
have observed greater success using primers
with less than 3000 matches.
At present, the software available for
primer design does not account for PCR
bias (2022). When faced with bias, it is
important to use additional tools to review
the ROI sequence, highlight the CpG and
non-CpG cytosines, and design adequate
primers. BioWord is a free Microsoft Word
plugin that allows manipulation, editing,
and processing of DNA sequences and has
proven useful for working with sequences
prone to PCR bias (23). Another available
option is a shareware version of the licensed
software FastPCR, which includes a tool for
in silico bisulfite conversion of non-CpG
cytosines (24).

PCR-based techniques

Bisulfite sequencing PCR

Bisulfite sequencing PCR (BSP) was the
first technique described for analyzing
DNA methylation status using PCR (25).
The technique consists of PCR amplifying
a bisulfite-converted DNA ROI, followed
www.BioTechniques.com

Review

Table 1. Comparison of commercially available kits for bisulfite conversion (single column format)
Provider
(References)

DNA Input

DNA Output (1)

Time
(2)

Additional notes

Link (3)

Reactions/
Cost (4)

Citations (5)
search Words

Zymo
DNA Methylation Gold Kit (D5005
& D5006)

500 pg 2 g
(Optimal 200
500 ng)

10 ml
>99%
>75%

Modified DNA can

be stored at
20C for to1 month

goo.gl/zAwu1

50 rxns (2.7)
200 rxns (2.3)

1.380
Cells-to-CpG-Bisulfite
Conversion

Qiagen
EpiTect Bisulfite Kit (59104)

1 ng 2 g

20 ml
>99%
NA

Protocol for FFPE

Samples

goo.gl/nNTeu

48 rxns (4.4)

1.020
EpiTect- Bisulfite-Kit
Qiagen

Zymo
DNA Methylation Direct Kit
(D5020 & D5021)

50 pg- 2 g
(Optimal 200 500ng)

10 ml
>99.5%
>80%

Protocol for FFPE

Samples

goo.gl/JJnDI

50 rxns (3.6)
200 rxns (2.5)

95
Zymo DNA Methylationdirect-kit

Zymo
EZ DNA Methylation-Lightning
Kit (D5030 & D5031)

100 pg - 2 g
(Optimal 200 500ng)

10 ml
>99.5%
>80%

For long-term use

store at -70C

goo.gl/zAwu1

50 rxns (3.6)
200 rxns (2.5)

0
Methylation Lightning-kit
Zymo

Epigentek
BisulFlash DNA Modification Kit
(P-1026050)

0.2 ng 1
g (Optimal
200500 ng)

>90%
>99.9%

2,5

Protocol for DNA

input 0.2 ng/50 cells

goo.gl/TU79j

50 rxns (2.2)

7
Epigentek BisulFlash DNA
Modification Kit

Chemicon (Millipore)
CpGenome Turbo Bisulfite
Modification Kit (S7847)

500 pg- 1 mg
(Optimal 1 ng
-1mg)

25 ml
>99.9%
NA

Modified DNA can

be stored at 20C
for to 2 months

goo.gl/xMNDz

50 rxns (3.3)

21
CpGenome Bisulfite
Modification-Kit-Millipore-fast

Chemicon (Millipore)
CpGenome Fast DNA
Modification Kit (S7824)

1 ng - 1 mg
(500 pg of DNA
can be used)

3545L
NA
NA

24

Modified DNA can

be stored at 20C
for to 2 months

goo.gl/3sern

Epigentek
Methylamp Modification Kit
(P-10011 & P-10012)

1 ng-1 g.
(Optimal
50200 ng)

818 L
99.5%
NA

Modified DNA can

be stored at 20C
for to 2 months

goo.gl/7ElOC

40 rxns (2.7)
80 rxns (2.5)

Epigentek
Methylamp DNA Isolation &
Modification Kit (P-100240)

1 ng-1 g.
(Optimal
50200 ng)

818 L
>99.9%
>90%

Protocol for adhesive

cells, plasma, serum,
and body fluids

goo.gl/IoOvy

40 rxns (5.0)

New England Biolabs

EpiMark Bisulfite Conversion Kit
(E3318S)

50 ng2 mg

40 ml

Modified DNA can

be stored at 20C
for up to 2 months

goo.gl/kKRLk

48 rxns (2.8)

Sigma-Aldrich
Imprint Bisulfite
DNA Modification Kit (MOD50)

10 ng to 1 mg
(Optimal 10 ng
- 1 mg)

820 L
>99%
NA

50 pg / 20 cells
protocol

goo.gl/IUhOI

Invitrogen
Cells-to-CpG Bisulfite Conversion
Kit (4445555)

50 pg-5g
(Optimal 100
ng - 1 g)

10 L-40
99.5%
NA

Protocol for blood,

10 - 105 cells (optimal
5000 -105 cells)

goo.gl/upJ1A

Human Genetic Signatures

MethylEasy Xceed
Human Genetic Signatures

50pg -5 mg
(12 L -100 L)

50l
>99,9
>90%

2,5

Protocol for input

equivalent to 8
human cells

goo.gl/4JuMI

4,5

Magneticbead
purification

goo.gl/QZ2JN

2,5

<50 ng input DNA

protocol

goo.gl/miigf

Diagenode
MagBisulfite Magnetic bisulfite
conversion kit (AF-1060024)
Clontech.
EpiXplore Methyl Detection Kit
(631968)

>1 ng
(Optimal 100ng
-1g)
50 pg5 mg

50l
>99%
NA
12100 ml
NA
NA

T25rxns (8.0)

50 rxns (4.3)

50 rxns (5.0)

17
CpGenome Bisulfite fast
Modification-Kit-Millipore
67
Methylamp DNAModification-Kit -CoupledDNA
1
Methylamp-Coupled
Epigentek
8
EpiMark BisulfiteConversion-Kit)
46
Imprint-DNA-ModificationKit Sigma
1
Cells-to-CpG-BisulfiteConversion-kit

40 rxns (6.5)

52
MethylEasy Xceed

24 rxns (13.6)

0
MagBisulfite + Diagenode

10 rxns (17.5)

EpiXplore-Methyl-Detection

1. Top: Output volume; Middle: Expected conversion efficiency; Bottom: Expected recovery. 2. Approximate time length of conversion process in hours.
3. Weblink shortened using Google URL shortener service. 4. Number of reactions for each kit and costs for reaction in U.S.-Dollars (in parentheses), derived from providers web page (currency conversion if required). 5. Number of citations found using Google Scholar (using the references as search words).
Substitute of Epigentek kit, old version no longer available. Abbreviation: rxns: Reactions. NA: No available information found online.

by Sanger sequencing of the product either

directly or after cloning into a suitable vector.
Direct-BSP: By comparing sequencing
results with the respective reference
genomic DNA sequences, direct sequencing
of PCR products provides information on
the average methylation status for each CpG
dinucleotide. Direct-BSP is the shortest
form of BSP, but holds several technical
challenges inherent in sequencing, such as
poor signal quality and artifacts in cytosine
signals that may affect electropherogram
analysis; it also has a low sensitivity (26).
Because of these difficulties, BSP with
cloning is more common.
Cloning-based BSP: In cloning-based
BSP, PCR products are cloned into a vector
and transformed into competent E. coli
Vol. 55 | No. 4 | 2013

cells. After expansion and purification of

the plasmids, the PCR product inserts are
sequenced. The CpG methylation status
for each CpG dinucleotide in the ROI is
determined by sequencing each expanded
clone (27,28). The resulting averages are
referred to as DNA-methylation haplotypes. Cloning-based BSP requires at least
six sequencing reactions to obtain a sensitivity higher than direct BSP (29), making
this an expensive and labor-intensive
option that is especially cumbersome for
population-based studies.
Digital (single-molecule) BSP: Another
BSP option for producing DNA-methylation haplotypes is digital-BSP (22,30).
This method requires serial dilution of a
DNA template to optimize conditions for

184

PCR amplification of a single converted

DNA molecule per reaction tube (via the
Poisson distribution), thus avoiding both
PCR bias and cloning. Digital-BSP is
considered the gold standard for detecting
the methylation status of specific loci (22).
However, this method is inefficient because
87% of the reactions cannot be analyzed,
and 3% are control reactions; thus, useful
information is only obtained from the
remaining 10% (22). An alternative
approach is to use MS-HRM to select the
clones for sequencing (31).
Primer design considerations: Methylation independent PCR (MIP) primers
should be designed to allow the amplification of bisulfite-converted DNA
regardless of methylation status. Primers
www.BioTechniques.com

Review

should also not bind regions containing

CpG dinucleotides (Figure 2A) (25) and
should flank a sequence of converted DNA
containing as many thymines originating
from the conversion of non-CpG cytosines
as possible (25). Guidelines for designing
BSP primers were initially published by
Clark et al. in 1994 (25).
Recently, several more techniques have
been developed using primers based on
the same principles (32). One variant of
the direct-BSP method uses two rounds
of nested PCR with primers designed by
standard methods for the first round and
primers with a GC-rich tag at the 5 end for
the second round. This primer modification
is intended to reduce non-specific amplification during direct-BSP and compensate
for the frequent artifacts seen in direct-BSP
results (33,34).
Bias in BSP: Several studies using MIP
primers have shown bias toward unmethylated or methylated alleles (8,20), likely due
to sequence differences between methylated
and unmethylated alleles (34). For example,
Warnecke et al. found a 33-fold amplification bias toward the unmethylated allele
when assaying a region of the RB1 gene
promoter (20). In some cases, adjusting
MgCl2 concentrations or redesigning the
primers to bind the opposite DNA strand
may be sufficient to resolve this bias (20,34).

Shen et al. found that, in some instances,

adjusting the annealing temperature
may correct this bias (35). Wojdacz et al.
developed a new approach to primer design
that allows the use of annealing temperature changes to adjust for amplification
bias (36,37). These new guidelines for bias
compensation are described in detail in the
MS-HRM section of this article.
Another potential source of error occurs
in cloning-based BSP methods. Cloning
biases may skew the reliability of results
generated from BSP assays (22,34). There is
evidence that amplicons without cytosines
may be more difficult to clone efficiently (8).
Although direct-BSP has low sensitivity, it
provides more accurate detection of differences as low as 20% in methylation status
in a single CpG (29).
Data analysis: Quantification of methylation levels is determined by comparing
the relative peak heights of cytosine and
thymine (or adenine and guanine in cases
of complementary strand sequences) (25)
in each CpG position in the electropherogram (Figure 2B). A qualitative analysis
of bisulfite sequencing results can be
performed if a clear single peak is present
for each CpG cytosine position. In that case,
a thymine peak would be interpreted as a
non-methylated CpG, and a cytosine peak
would represent a methylated CpG. Analysis

of raw sequence data from direct-BSP is

often difficult, but correction algorithms
aid data interpretation. The Epigenetic
Sequencing Methylation Software (ESME)
program includes an algorithm to analyze
direct-BSP sequencing results and provides
a quality control filter (Table 4) (29).
ESME may also be used to analyze
cloning-based BSP electropherograms (29).
In cloning-BSP or digital-BSP, satisfactory
sequencing results belonging to the same
sample should be averaged to determine the
level of methylation for each CpG position.
This task is facilitated by BiQ-Analyzer and
BISMA (38,39) (Table 4).
BSP selection: Different BSP methodologies are optimal for different methylation studies, depending on the particular
conditions of a study and other parameters, including cost, research question, and
available samples. For example, the study
initially validating digital MethyLight
is a case where digital-BSP was the best
choice, since it allowed accurate validation
of another single-molecule technique and
the use of automated PCR-processing for
a large number of sequencing reactions
(30). In many other cases, cloning-BSP is
preferred because it is the only option for
determining DNA-methylation haplotypes
in general laboratories (34). Direct-BSP
was selected to assess DNA methylation

Table 2. Comparison of commercially available kits for DNA methylation controls

Provider (Reference)

Millipore
S8001
(S8001M & S8001U)

Millipore
Chemicon-CpGenome
Universal Methylated /
Unmethylated DNA
(S7821 & S7822)
Qiagen
Human control DNA set
(Converted methylated
and unmethylated
and unconverted
unmethylated)
(59695)
Clontech
Unmethylated HCT116
DKO gDNA 10 g 3521
EpiScope Methylated
HCT116 gDNA 10 g

Amount / Cost (1)

Features

Notes

5g / 20L
$ 212

Methylated DNA
HCT116 DKO cells DNA methylated by
M.SssI DNMT (EC 2.1.1.37)
>95% of CpGs methylated
Non-Methylated DNA
HCT116 DKO cells DNMT1 (-/-)
DNMT3b (-/-)
<5% of CpGs methylated

Storage: -15C to -25C in aliquots

protected from freeze-thaws for up
to 2 years
Do not freeze and thaw the same -20C
aliquot more than 3 for best results
Storage at -70C for long-term use

Methylated DNA
Enzymatically methylated human male
genomic DNA

Storage: -15C to -25C in aliquots

protected from freeze-thaws for up
to 2 years
Do not freeze and thaw the same
-20C aliquot more than 3 (-70C for
longer storage)

goo.gl/tkm8z
goo.gl/AYQ8A

238

Non-Methylated DNA
NA
HCT116 DKO Gdna
DNMT3b (-/-)
DNMT3a (-/-)
Methylated DNA
In vitro methylation of HCT116 gDNA

The control non-converted DNA is

shipped at room temperature and
should be stored at 20C immediately
upon receipt (at least 6 months without
reduction in performance)
Bisulfite converted DNA is stored in EB
Buffer in a ready-to-use 10 ng/ml solution

goo.gl/D9z8K

53

3520 EpiScope Methylated HeLa

gDNA

goo.gl/RsCTk

10 g
$ 299

100 L each of a 10 ng/l

solution
$ 358

$153.00
10 g

Methylated DNA
In vitro methylation of the control DNA
using SssI methylase
Non-Methylated DNA
NA

Link (2)

goo.gl/6DMnf
goo.gl/tFRPk

Citations (3)

67
40

D5014
Methylated DNA
5+5 g/20+20 L ($ 402)
HCT116 DKO cells DNA enzymatically
D50141
methylated by CpG Methylase
Recommends using hot-start DNA
40
5 g/20 L
Non-Methylated DNA
polymerases for amplification of
goo.gl/jZg6L
33
($ 223)
HCT116 DKO cells DNMT1 (-/-)
bisulfite-treated DNA
16
D50142
DNMT3b (-/-)
5 g/20 L
<5% of CpGs methylated
($ 223)
1. Cost in U.S. Dollars from providers web page. 2. Weblink shortened using Google URL shortener service.
3. Number of citations found using Google Scholar. NA: No available information found online. Qiagen portfolio of controls includes additional choices, however the
mentioned 59695 is a set that includes the other references (59655, 59665, 59568).
Zymo
Human Methylated &
Non-methylated DNA
Set DNA / primers
(D5014, D50141,
D50142)

Vol. 55 | No. 4 | 2013

186

www.BioTechniques.com

BSP assay

Figure 2.

Review

B
Classical primer design for BSP

Cloning-based BSP
CpG1

CH3

CH3

CpG2

CpG1

CpG1

CpG2

Y (30%)

Y (60%)

Y (20%)

Y (50%)

CGNNNNTTNUNNUNUNNCGUNCGTN

CGNNNNTTNCNNCNCNNCGCNCGTN
------------------------------

CpG2

Post-bisulfite sequence binding region

Pre-bisulfite sequence binding region

CH3

Direct BSP

--------------------------------

Methylated DNA

Methylated DNA

UGNNNNTTNUNNUNUNNUGTNUGTN

CGNNNNTTNCNNCNCNNCGCNCGTN
-------------------------------

--------------------------------

Non-methylated DNA

Non-methylated DNA

Y (50%)

Forward Primer: NNNNTTNTNNTNTNN

Reverse Primer : NNANANNANAANNNN *

C: Methylated (100%)
T: Non-methylated (0%)

Y (80%)

MSP assay

CC

MSP Primer design

D
ElectrophoresisPossible results

Methylated DNA
CH3

CH3

CH3

NNCGNNCNCNNCGCNCG

Pre-bisulfite DNA sequence

NNCGNNCNCNNCGNNCG

Pre-bisulfite DNA sequence

CH3

CH3

CH3

NNCGNNUNUNNCGUNCG

Methylation
level
100%

NNCGNNTNTNNCGTNCG

Methylation
level
0,1%-99,9%

Methylation
level
0%

Post-bisulfite sequence amplicon Forward primer design

Non Methylated DNA
NNUGNNUNUNNUGUNUG

NNTGNNTNTNNTGTNTG

Post-bisulfite sequence amplicon Forward primer design

AV
rA

ZFN

Figure 2. Primer design and results for bisulfite specific PCR (BSP) and methylation specific PCR (MSP). (A) BSP primer. The dashed line indicates the sequence of the primer binding region in this example. Note that CpGs are avoided in the design of this kind of primers. *For simplicity, we use the same binding sequence of the forward primer to illustrate a hypothetical reverse primer design. (B) Simplified electropherogram schema of possible results from BSP
for two CpG cytosines. Left panel: Cloning-based BSP. Possible sequencing results for a single clone. The possibilities are reduced to methylated (remains
C on the sequencing data) or unmethylated (T replaces C in comparison with non-treated sequence). Right panel: Direct BSP C denotes methylated status
(100%), T denotes non-methylated (0%) and Y (C + T) denotes different methylation percentages. (C) Chart of an example of MSP primer design over a binding site specific for methylated DNA (top) and non-methylated DNA (bottom). The untreated sequence (left) is modified depending on its DNA methylation
status (middle). This sequence is used to design the forward primer by substituting the Us with Ts (D) Diagram of the possible results of an MSP assay on an
electrophoresis gel. N: non-affected nucleotides in bisulfite treatment. The Us and Ts in red represent the uracils derived from bisulfite conversion of CpG cytosines and the corresponding thymines in the primer sequence. The CpG dinucleotide and the corresponding UpG or TpG sequences are in bold and underlined.

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in BDNF to complement initial results

from MSP screening experiments (40).
Before deciding between direct-BSP and
cloning-BSP for a particular application,
we recommend testing and comparing
previously validated primers and strategies
(26,27,33).
Methylation Specific PCR
Methylation specific PCR (MSP), first
described by Herman et al. in 1996,
determines the methylation status of an
ROI through selective amplification of
methylated and unmethylated alleles. The
two-tube approach employs two primer sets:
one binding specifically to the methylated
sequence and another binding to the
unmethylated sequence (11,41) (Figure 2C).
A two-round variant of MSP, referred to as
nested-MSP (N-MSP), has been described
and can be used in special cases (42).
MSP is a simple method that requires
resources commonly available in a molecular
genetics laboratory and, once standardized,
is effective for detecting methylated or
unmethylated alleles without quantification.
Processing up to 24 samples for both primer
sets using conventional MSP requires about
4 h. Commercially available PCR master
mixes for MSP (EpiTect MSP Kit, Qiagen,
Hilden, Germany) are available; however,
only conventional PCR reagents, including
Hot-Start Taq polymerase, are required for
the setup of MSP (43). While MSP assay
kits are not commercially available, the
MethPrimerDB database is available for
help in selecting MSP primers (44).
Several real-time PCR adaptations of
MSP also have been developed, including
MethylQuant, a common option based on
the measurement of increased fluorescence
from SYBR Green I (45), and a real-time
MSP approach combining conventional
qPCR measurements with an additional
melting step to detect amplicons associated
with incomplete DNA conversion (46)
or to distinguish the methylation status
of individual alleles by comparison with
standards of known allelic methylation

status for an SNP located in the amplicon

region (47).
Primer design considerations: As
described by Herman et al., both methylated
and unmethylated MSP primer sets should
be designed to anneal to the same CpG
containing region. MSP primers should
include abundant CpG sequences at the
primer binding sites to provide maximal
discrimination between the methylated and
unmethylated alleles. For the same reason,
these CpGs should be as close as possible to
the 3 region of the primer (11) (Figure 2C).
Additionally, a high number of thymines
derived from non-CpG cytosines should be
included to ensure specificity for converted
DNA. MSP primer design is facilitated by
the software listed in Table 3.
Data analysis: An amplification product of
the correct molecular weight on an electrophoresis gel can be interpreted as methylated
or unmethylated, depending on the specific
primers used (11). The presence of amplification products using both sets of primers
indicates a sample with both methylated
and unmethylated DNA in the ROI (Figure
2D). However, a band from a reaction
with methylated-specific primers might
be a false positive. To avoid misinterpretation, inclusion of unmethylated DNA,
non-converted DNA, and no-template
negative controls is required (46,48).
Likewise, the absence of an amplicon could
be due to issues with the PCR reaction and
must be controlled for as well (49).
The primary limitation of this technique
is that it is qualitative (11). In general,
well-standardized MSP assays provide
information restricted to three possible
outcomes: (i) presence of a methylated allele,
(ii) presence of an unmethylated allele,
or (iii) presence of both alleles. In assays
intended to test MSP sensitivity, several
ratios of the methylated and unmethylated
DNA were used as templates. The results
showed no clear correspondence between
band intensity and dilution ratio, with
many cases exhibiting very similar bands
even for disparate levels of DNA methyl-

ation (50). On the other hand, several

MSP assays demonstrated high sensitivity,
detecting methylation percentages (MP)
as low as 0.1% (50 pg of methylated DNA
out of 50 ng of total DNA) or 1% (0.1 ng
of methylated DNA out of 10 ng of total
DNA) in different studies (11,43,50).
Possible challenges: Low quality DNA is
associated with a decrease in reproducibility
(51). As mentioned above, it is critical to
avoid amplification of non-converted DNA
using MSP primers (11,52). Kristensen et
al. identified false positive MSP results due
to incomplete bisulfite-conversion, which
is particularly problematic if only four or
fewer non-CpG cytosines are included in
the primer binding region (52). This issue
has been associated with the apparent low
reproducibility of numerous MSP assays
(46,53). On the other hand, even after PCR
amplification, MSP results can be validated
by means of pyrosequencing to confirm
the full conversion of every non-CpG
cytosine (49). In MSP, PCR for methylatedspecific or unmethylated-specific primer
sets can frequently be standardized with
non-identical PCR conditions (for example,
different annealing temperatures) (11),
possibly through inherent differences in
sequence composition between primer
sets. Therefore, identical PCR conditions
for both MSP primer sets are not required
for accuracy (11).

Real time PCR-based methods

MethyLight
Dual TaqMan labeled probes were developed
for genotyping studies several years ago
(54). Eads et al. subsequently introduced
the use of TaqMan technology to determine
DNA methylation status in specific genomic
regions, a technique that was named MethyLight (55). Peter Lairds group defined four
types of MethyLight reactions, depending
on which oligonucleotides are designed to
discriminate the methylation status: (i)
only the primers, (ii) only the TaqMan
probe, (iii) both primers and probe, or (iv)

Table 3. Freely available software for primer design for methylation analysis
In silico PCR
on bisulfite
treated DNA

Tool

MSP /BSP
design

Design for bias

compensation

Primer thermodynamic evaluation

Additional
advantage

Additional disadvantage

Bi-Search

yes

no

yes

Able to test the

primer specificity

It does not inform the cause of primer

rejection in in-silico PCR

yes

no

no

Simple and
flexible

No automatic function to search for primers

reverse complementary to the input sequence

no

Methyl Primer Express

(Applied Biosystems)

yes

no

no

No very intuitive

no

www.expbiosystems.com

Perl primer

yes

no

yes

Many customable
features
Customable CpG
island definition

Bugs on the copy-paste function

no

perlprimer.sourceforge.net/

yes

no

yes

User registration is necessary

No

www.mspprimer.org

MethPrimer

MSPPrimer

Vol. 55 | No. 4 | 2013

Primer design with

higher specificity
Nested MSP
design included

188

yes

Link
bisearch.enzim.hu
www.urogene.org/methprimer/

www.BioTechniques.com

Review

Table 4. Multi-purpose software for DNA methylation test design and analysis of results.
Tool

Method

BioWord
FastPCR
MethGraph
MethBlast

For all PCR-based

DNA methylation
techniques

Function

Web link

Editing, replacing and assistance for primer design

http://sourceforge.net/projects/bioword/

In-silico analysis of several designed primers, bisulfite conversion of nonCpG cytosines

www.biocenter.helsinki.fi/bi/programs/fastpcr.htm

Automatic genomic representation of the designed primers

http://mellfire.ugent.be/methgraph/

In-silico evaluation of oligonucleotide sequence similarities to bisulphite

modified genome sequences
Generate possible assays for the supported experimental methods in the
ROI

http://medgen.ugent.be/methBLAST/methBLAST_cs.php

MethMaker
Beacon
Designer
software
ESME

MethyLight

Primer/probes design and primer evaluation

Direct BSP

Analysis of results

www.epigenome.org/index.php?page = download

BiQ Analyzer

Cloning-based BSP

Analysis of results

http://biq-analyzer.bioinf.mpi-inf.mpg.de/download.php

BISMA

Cloning-based BSP

Analysis of results

www.premierbiosoft.com/molecular_beacons/index.html

http://biq-analyzer.bioinf.mpi-inf.mpg.de/download.php

Poland
MELT

http://methmarker.mpi-inf.mpg.de/

www.biophys.uni-duesseldorf.de/local/POLAND//poland.html
MS-HRM

Tm Calculation for PCR amplicon

OligoCalc

http://web.mit.edu/osp/www/melt.html
www.basic.northwestern.edu/biotools/oligocalc.html

The software displayed in this table are freely available online with two exceptions:
Shareware with full functionality available only for a limited time.
Licensed software, demo version with limited functionality.

none (in cases where a control reaction is

required to discriminate the converted
DNA) (55) (Figure 3A). Using these
reactions, several variations of MethyLight have been proposed to address
different biological questions, such as the
amount of methylated versus unmethylated alleles (56) or methylation status
at the CpG dinucleotide level (which
would be very expensive) (55). The most
commonly used MethyLight methodology uses two primers and a TaqMan
probe designed to bind the methylated
allele specifically and requires a reference
gene for normalization (55) (Figure 3A).
It is important to note that MethyLight,
depending on the method subtype, can
assess the methylation status of all CpG
sites covered by the TaqMan probes.
The quantitative aspect of MethyLight has been explored since its
inception. Analyses using different
ratios of methylated to unmethylated
DNA have been employed to verify the
linearity of this quantitative assay, with
a high linear correlation found between
the dilution ratios and the MethyLight
MP measurements (55,57). MethyLight
has shown higher levels of accuracy
and lower rates of false negatives when
compared with previously described
techniques (45,55). For this reason,
MethyLight is frequently used to validate
other techniques for DNA methylation
studies (36). However, unlike MS-HRM
(36), the most commonly used MethyLight technique cannot detect heterogeneous methylation in a sample because
the MethyLight primers and probes are
designed to measure a specific methylation pattern (fully methylated).
Primers and probe design considerations: In a MethyLight assay, it is necessary
Vol. 55 | No. 4 | 2013

to normalize each qPCR reaction using sets

of primers and probes that bind a converted
DNA region independent of its methylation
status (Figure 3A, top). Therefore, each
MethyLight assay should include both an
ROI amplification reaction and an MIP
reaction for the control region. Currently,
commercially available Beacon Designer
software (Primer Biosoft, Palo Alto, CA)
is the method of choice for MethyLight
primer design. This program is able to design
MethyLight assays, but in practice it is
restricted to CpG islands, possibly because
CpG density in the island shores and in
some promoters lacking islands is low.
Data analysis: Depending on the MethyLight design, relative fluorescence units
(RFUs) should be used to calculate the
methylation percentage. For the common
MethyLight design described in this section,
the broadly accepted determination formula
for DNA methylation percentage is shown
in Equation 1 (55).
In order to evaluate the methylation
status of an ROI using MethyLight, it is
best to select a previously validated primer/
probe set for that ROI if a similar research
question is to be addressed. Houshdaran et
al. (58) have performed ~300 MethyLight
assays and have made the primer and probe
sequences available.
Example of MethyLight selection: MethyLight is the technique of choice when
a study requires accurate quantitative
assessment of DNA methylation. It has
been used in a number of cancer associated
DNA methylation studies, including the
development of an assay to measure the
presence of methylated alleles in three genes

associated with colorectal cancer (59). Here,

the assay was focused on clinical applications of cancer detection. It should be noted
that the cost of using TaqMan probes can be
higher than other real-time PCR methods
that utilize cheaper intercalating dyes (46).
This is of particular importance if the
sample size of the proposed study is large,
or if a significant number of ROIs is to be
assessed.
Methylation-sensitive high
resolution melting
In the DNA double helix, a cytosine and
a guanine of complementary strands are
linked by a triple hydrogen bond while
a thymine and an adenine are joined by
a double hydrogen bond (36). Therefore,
base composition can directly influence
the thermodynamic behavior of DNA in a
melting analysis. Tm is defined as the temperature at which the PCR product dissociates
into two single strands and a sharp drop in
fluorescence of a DNA intercalating dye is
observed (37). This basic principle can be
used to discriminate between methylated
and unmethylated alleles following bisulfite
conversion. Distinction between alleles
is achieved through Tm analysis of the
MIP-PCR products in the ROI, in which
the methylated allele usually has a higher Tm
than the unmethylated allele (60) (Figure
3D).
Initially, Worm et al. (61) described an
in-tube melting protocol for analyzing DNA
methylation prior to the development of
high resolution melting (HRM) technology.
After technical improvements in melting
assays, Wojdacz et al. developed a DNA

Equation 1.
ROI signal of methylated status (Sample)
Target ROI gene Methylated STATUS (Fully methylated standard)
MIP (DNA input amount normalizing) (Sample) / MIP input amount normalizing gene (Fully methylated standard) x100

190

www.BioTechniques.com

Review

MethyLight Assay

BB

MIP primers on the

evaluated sample

Fluorescence

MIP primers on
100% Methylated DNA
ROI primers on
100% Methylated DNA

ROI primers on
the evaluated sample

Methylated CpG
Non-Methylated CpG
Fluorescent dye extreme of MethyLight probe
Quencher dye extreme of MethyLight probe

Cycle

Fluorescent dye extreme of MethyLight probe (DNA amount

normalization-reaction)
Fluorescent dye extreme of MethyLight probe (reactions for
non-methylated DNA)

MS-HRM Assay

D
0 % Methylated DNA

Example of amplicon between primers

Methylated Standard

-d(RFU)/dT

2500

Pre-bisulfite amplicon sequence

NCGTTCGNNCNNC NCGNCGCNCGTN
G G G G
G
NGCAAGGCNNANNANGCNGCAN
GCAN

1500

500

76

78

80

Temperature (Celsius degree)

Post-bisulfite amplicon sequence

From Methylated DNA

NCGTTCGNNTNNTNCGNCGTNCGTN

G G
G
NGCAAGGCNNANNANGCNGCAN
GCAN

From Non-Methylated DNA

NTGTTTGNNTNNTNTGNTGTNTGTN
ACAAGACNNANNANGCNGCANGCAN
A A
A
NG

Relative Signal Difference

70

100%
90%

60

80%
50

70%
60%

40

50%
30

40%
30%

20
10

20%

Evaluated
sample

10%
0%

76

78

80

Temperature (Celsius degree)

methylation assay implementing HRM

technology: the methylation-sensitive
high-resolution melting (MS-HR M)
techniques (36). The MS-HRM methodology consists of real-time PCR using
bisulfite-converted DNA (regardless of the
methylation status) and melting analysis
of PCR products (HRM) to discriminate
the ROI methylation status reflected in the
thermodynamic behavior of the MS-HRM
amplicon.
The MS-HR M method enables
assessment of the percentage of the
methylated allele present for a particular
sample in an ROI. This is possible through
comparison with melting standard curves
created by different dilution ratios of
methylated and unmethylated DNA
controls (37). Since the technique analyzes
the melting properties of the final PCR
products, MS-HRM not only evaluates
fully methylated alleles in proportion to
Vol. 55 | No. 4 | 2013

fully unmethylated ones, but is also able to

detect heterogeneously methylated samples
(62).
PCR bias: As for other MIP based amplifications, potential PCR bias for MS-HRM
was evaluated during development of the
technique (21). MS-HRM showed a strong
amplification bias toward unmethylated
sequences when the classic recommendations for primer design stated by Clark et
al. were followed (25). In contrast, using
the recommendations of Wojdacz et al.
for primer design (21), variations of the
annealing temperature in the PCR cycling
step allowed for control of PCR bias (21).
Monitoring of real-time PCR amplification
establishes an additional quality control step
for MS-HRM experiments (13). Similar to
digital-BSP, digital MS-HRM is also useful
for reducing PCR bias (62). Considering
the possibility of PCR bias, it is important
to highlight that quantitative methyl-

191

Figure 3. Real time PCR assays: MethyLight

and MS-HRM. (A) Schematic lollipop graph
of MethyLight subtypes. The most used
approach consists of primers and probes
designed for converted methylated DNA sequences and uses MIP primers to normalize the DNA input (top). Another choice is to
design a set of primers and probes specific
for methylated DNA and another set specific
for non-methylated DNA (middle). In these
cases, the sum of both signals is used to
normalize the individual signals. Similar to
classic MSP, one primer set is specific for
methylated DNA and the other set for unmethylated DNA (bottom). One probe, designed to bind DNA independent from its
methylation state, is used with both primer
sets. The normalization procedure is similar
to the one for the middle section. (B) Amplification curve of the most used MethyLight
subtype (top in A). The RFU value determination for 100% methylated control DNA allows calculation of the percentage of methylated molecules in the evaluated sample.
The MIP signals allow an adequate control
of DNA input amount for both the evaluated
sample and 100% Methylated DNA. (C) Simplified outline of an MS-HRM amplicon for
the analysis of methylation status. The triple
hydrogen bond of G and C is represented.
(D) On top: schematic graph of the negative
first derivative of the melting-curve. DNA
that is 0% methylated has a lower melting
temperature peak in comparison to 100%
methylated DNA. Non-converted DNA has
the highest number of triple hydrogen bonds
and therefore presents the highest melting
peak. On bottom: schematic plot of the differences for the normalized signal of the
standard curves. The plot also presents a
curve for an illustrative sample that is located between the 10% and 20% standard dilution curves. (In this type of melting curve,
0% of methylation is used as the cluster
of reference.) Conventions as in Figure 2.

ation analysis with MS-HRM is based on

the assumption that methylation levels of
CpG sites between the primers is the same
as methylation levels of CpG sites covered
by the primers.
Primer design considerations: MS-HRM
primer design follows the same general
principles of classic MIP design as previously detailed by Clark et al. (25). However,
in order to compensate for PCR bias, there
are new recommendations for MS-HRM
primer design that advise inclusion of one
or two CpG annealing sites (located as far
as possible from the 3end of the primers to
avoid methylation specific amplification)
(Figure 3C) (60). Currently, there are no
programs for MS-HRM primer design that
incorporate the new recommendations to
compensate for PCR bias. Finally, several
programs such as OligoCalc, Poland, and
MELT (Table 4) can predict the melting
curves of the PCR products.
www.BioTechniques.com

Review

Table 5. Comparison of PCR-based DNA methylation techniques

Basics
MIP primers to amplify the region of interest (ROI)
(for the population of cells that conform the
sample) and sequencing

Advantages
Information of methylation at CpG resolution level
One sequencing reaction for sample

Disadvantages
No information at single molecule level
Possible PCR bias (no detectable)
Considerable noise in sequencing results

Cloning-based BSP

MIP primers to amplify the ROI (for the population

of cells that conform the sample), cloning and
sequencing reaction for each clone

Information of methylation at CpG resolution level

Information at single molecule level
Reduced noise in the sequences reaction

PCR bias
Cloning procedure (time and money consuming)
PCR bias (no easily detectable)
Possible cloning bias (no detectable)
At least 5 sequencing reactions for sample

Single molecule
(digital) direct BSP

MIP primers to amplify the ROI using serial dilutions of

template to obtain single molecule PCR products
Sequencing the positive amplifications to obtain a
number of single molecule sequences

Quantitative information of methylation at

CpG resolution level
Information at single molecule level
Reduced noise in the sequences reaction
No PCR bias
No cloning bias

Serial elusion step to determine the most effective

input for each PCR reaction
At least 25 sequencing reactions for sample for
acceptable sensitivity
5 fold increase in sequencing reactions for one
sequencing result

Classical MSP

MSP primers, PCRs and electrophoresis

CpG site at or near the 3 primer allow
amplification of only methylated or
unmethylated DNA
Stringent annealing temperatures to avoid
amplification of unconverted DNA

High sensitivity
May be very cost-effective in settings where
the objective is detection of any degree of
methylation at primer binding region
No sequencing reaction required

Low specificity
Not quantitative
False-positive results (no easily detectable)
Two separate tube reactions for assay
No information of methylation at CpG resolution
level neither at single molecule level

Real Time Based

MSP

MSP primers
CpG site at or near the 3 primer allow
amplification of only methylated DNA or
unmethylated DNA
Stringent annealing temperatures to avoid
amplification of unconverted DNA

Feasibility of a semiquantitative approach

Very high sensitivity
Information of methylation at primer binding region
No sequencing reaction

MS-HRM

MIP / primer set for compensation of PCR bias

One step PCR and HRM

High sensitivity, a quantitative approach

One primer set in a closed tube assay
Information of methylation at regional level
Immediate detection of false-positive amplification
and PCR bias

No quantitative (semiquantitative approach)

and low specificity
Two primer sets, each set used in one separated tube
reaction (possibility of different amplification
efficiencies)
No information of methylation at CpG resolution level
No information at single molecule level, unless using
digital approach
No information of methylation at CpG resolution
level
No information at single molecule level, unless
using digital MS-HRM

MethyLight

Different kinds of Methylight approaches.

Refer to text for particular advantages and
disadvantages of each kind of MethyLight

Very high sensitivity

Closed tube assay
Quantitative approach

Direct BSP

biotechniques_add.pdf

9/16/13

10:39 AM

Data Analysis: Wojdacz et al. (36,37)

proposed a method for estimating
methylation levels by comparing the
melting patterns of standard templates
with known proportions of methylated
and unmethylated DNA controls to the
melting patterns found in a sample. The
semiquantitative estimate is based on
similarities in HRM patterns without a
mathematical approach for calculating
the DNA methylation percentage. More
recently, Tse et al. (2011) implemented
an MS-HRM approach to quantify the
methylation status of each sample with
high reproducibility. Peak-height and area
under-the-curve from the normalized,
temperature-shifted difference curves
were used to generate linear standard
curves (13) (Figure 3D). Quantitative data
were obtained by interpolation of the first
derivative of the normalized melt curves,
generated by the linear regression analysis
of the standard curve (13). When heterogeneous DNA methylation patterns
are present in a sample, HRM analysis
will identify such heterogeneity by the
complex shape of the melting curves;
however, in such cases quantitative HRM
measurement is not possible (62). The
presence of SNPs in the amplicon region
could generate additional variations in
the melting profiles (37).

CM

MY

CY

CMY

Vol. 55 | No. 4 | 2013

192

No information at single molecule level unless

using digital MethyLight MethyLight
No information of methylation at CpG resolution
level, unless use of CpG specific probe

Examples of MS-HR M selection:

MSP-based assays only evaluate DNA
methylation for CpG sites present in the
primer binding region (usually <25 bp per
primer). In contrast, MS-HRM evaluates all
of the CpGs banked by the primers (usually
>80 bp), regardless of the methylation
status of CpGs within the primer binding
site (36). Therefore, MS-HRM provides
the ability to evaluate a larger genomic
region when compared with MSP-related
techniques (Figure 4) (11). MS-HRM is
a good choice for quantitative determination of DNA methylation levels, when
sequence level detail is not required (63). A
good example of the usage of MS-HRM is
a colorectal cancer study where the authors
distinguished different stages of the disease
and their correlations with the quantity of
DNA methylation (64).
Proper controls for PCR-based
DNA methylation analysis
In addition to the controls used in conventional PCR assays, other steps should
be taken to verify the accuracy of DNA
methylation data generated in PCR-based
assays. Unconverted genomic DNA is an
essential control that should be included in
all optimization processes for PCR-based
DNA methylation assays; it provides
information on the amplification of
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Figure 4.
Review

CpG coverage of PCR-based DNA methylation techniques

BSP assay

Evaluated CpGs

Non-evaluated CpGs

MSP assay

MethyLight assay

Fluorescent dye

Quencher dye

MS-HRM assay

Figure 4. CpG coverage of PCR-based DNA methylation techniques. A simplified lollipop schema
shows the CpG dinucleotides as circles. The methylation status is not represented. Grey circles
represent the non-evaluated CpGs in each corresponding technique. Red circles represent the
CpG dinucleotides evaluated in each method. In the MethyLight assay, the green oval represents
the fluorescent molecule and the black oval represents the quencher molecule.

non-converted DNA with primers that are

specific for the converted DNA. For specific
assays, amplification from bisulfite-treated
DNA should show a clear difference from
any possible result using non-converted
DNA.
In one of the pioneering MSP studies,
Herman et al. verified primer specificity for
the bisulfite modified p16 sequence using
untreated DNA in reactions with either
methylated-specific or unmethylatedspecific primers (11). As expected, no amplification was found when non-converted
DNA was used as a template. Nonetheless,
several reports of MSP standardization did
not include or report this kind of control
(65,66).
Similarly, the use of non-converted
DNA is also recommended when using the
MS-HRM technique during assay optimization (37). This type of control is the easiest
to include but, paradoxically, is the controal
most commonly omitted or not reported
(63). It allows experimental verification of
the specificity of the assay for converted
DNA. In most cases, there should be no
amplification products; however, in some
instances products will be amplified that
can be easily identified when compared with
the converted DNA (37).
Use of fully methylated and unmethylated DNA is a critical experimental
control as well. It should be noted that
Vol. 55 | No. 4 | 2013

DNA considered to be fully unmethylated

comes from a variety of different sources.
The practice of using DNA obtained
from peripheral blood mononuclear cells
(PBMC) as a fully unmethylated DNA
control is valid in cases where the samples
are indeed completely unmethylated at
the loci of interest. Several reports have
focused on detecting DNA methylation
status in peripheral blood, showing biologically important methylation levels for
multiple genes (52,67). For example, low
level methylation of many cancer-relevant
genes may be found in the PBMCs from
normal individuals. Therefore, the indiscriminate use of DNA from PBMCs as a
negative control in sensitive assays for DNA
methylation detection may be particularly
problematic (52).
Manufacturers of commercially
available DNA controls have different
strategies for providing fully methylated
and unmethylated DNA. For example,
fully non-methylated kits from Zymo and
Millipore use DNA from cells that contain
genetic knockouts of 2 key DNA methyltransferases, thus reducing methylation
levels by more than 95% (68).
Fully methylated DNA can be obtained
from M.SssI-methylated DNA from, among
many sources, double knockout cells for
DNMT1 and DNMT3b (Table 2). Another
alternative is to use the product from whole

194

genome amplification (WGA) with kits

such as REPLI-g (Qiagen), which does not
reproduce the DNA methylation pattern
and has a theoretical methylation level of
less than 10-6. However, this amplification
approach may carry the risk of reduced
representation of the loci of interest (69).
Therefore, the use of identical amounts
of methylated and unmethylated controls
derived from the same class of template
(genomic DNA or WGA-products) could
guarantee that equivalent amounts of
effective templates are included.

Discussion
Studying DNA methylation for a candidate
ROI using PCR-based methods is a topic
of present and future importance. There
are many advantages of genome-wide
platforms; however, PCR-based techniques
permit detailed analysis of specific regions
of the genome, including CpG island
shores. In addition, the associated costs of
implementing and executing PCR-based
techniques are lower, allowing the initial
study of several candidate ROIs. PCR-based
approaches also offer the advantage of a
lower burden of false discoveries and the
ability to confirm a large number of ROIs
identified in genome-wide screening of a
few samples (70).
DNA methylation analysis using pyrosequencing is a quantitative approach that
does not require a cloning step, but presents
the risk of PCR bias, similar to BSP. More
importantly, pyrosequencing instrumentation is not commonly available in a general
laboratory. For interested readers, comparisons and discussions of pyrosequencing
techniques are available elsewhere (71).
This article highlights several considerations for PCR-based DNA methylation
studies. The approaches reviewed here have
different advantages and disadvantages that
should be evaluated before starting any
DNA methylation study. Similarly, it is clear
that the different PCR-based techniques
discussed here have differences in CpG
coverage and possibility for quantitation
(Figure 4). A comparison of all PCR-based
DNA methylation techniques is presented
in Table 5.
Several considerations concerning
PCR-based methods for DNA methylation
analysis will be crucial for the consolidation
of the field of molecular epigenetics. Current
needs in this field include (i) detailed experimental comparisons of results obtained with
different PCR-based techniques (72), (ii)
the availability of a large number of predesigned PCR-based DNA methylation
assays to facilitate broad use, (iii) the implementation of minimum reporting guidewww.BioTechniques.com

Review

lines for manuscripts describing results of

PCR-based analyses of DNA methylation,
including details of experimental conditions such as controls, primer sequences,
and programs used for primer design (73),
(iv) the further development of additional
PCR-based techniques that allow DNA
methylation measurements in a more
quantitative and reproducible way (5), and
(v) the implementation of automatic and
multiplexed protocols for DNA methylation using currently available techniques
to improve efficiency and reduce costs (59).
For readers interested in genome-wide DNA
methylation analysis, we recommend two
available review articles (74,75). Finally, it
is critical to keep in mind that the results
of PCR-based DNA methylation methodologies are reliable only in an experimental
setting with adequate methodological
controls.

Acknowledgments

This work was supported by grants from

Colciencias (Contract # 401-2011),
UAN-VCTI, and UNAL-DIB. HGH
is a recipient of a PhD fellowship from
Colciencias. The authors would like to
thank the anonymous reviewers for their
important comments and suggestions.

Competing interests.

The authors declare no competing interests.

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Received 07 November 2012; accepted 10 September 2013.
Address correspondence to Diego A. Forero,
Laboratory of NeuroPsychiatric Genetics,
Biomedical Sciences Research Group, School of
Medicine, Universidad Antonio Nario, Bogot,
Colombia. E-mail: diego.forero@uan.edu.co
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biotechniques@fosterprinting.com

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