Trends in Analytical Chemistry 72 (2015) 114–122
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Trends in Analytical Chemistry
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t r a c
Development of techniques for DNA-methylation analysis
Li Zhang, Yu-Zhi Xu, Xiao-Feng Xiao, Jun Chen, Xue-Qin Zhou, Wen-Yuan Zhu, Zong Dai *,
Xiao-Yong Zou **
School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou, 510275, China
A R T I C L E
I N F O
Keywords:
Bioaffinity reaction
Biological recognition
Bisulfite treatment
Chemical cleavage
Determination
Direct oxidation
DNA methylation
DNA-methylation analysis
Enzymatic digestion
Epigenome research
A B S T R A C T
DNA methylation is an important mode of epigenetic modification and has great significance in biochemistry, medicine and genomics. The development of techniques for DNA-methylation analysis is
fundamental and pivotal for epigenome research. In around 30 years of diligent study, direct and indirect approaches were developed. Direct approaches mainly focus on global methylation analysis, while
indirect approaches rely on specific recognition of DNA-methylation loci. In this review, we introduce
typical methods, especially those based on developing the method of methylation-loci recognition from
the most used, bisulfite conversion, biological recognition, and the newly proposed chemical cleavage.
Furthermore, we assess the perspectives on the research methodology of DNA-methylation analysis. To
date, there have been few summaries of the research methodology of DNA-methylation analysis. The review
is envisioned to be helpful for the researchers to design novel methods of DNA-methylation analysis.
© 2015 Elsevier B.V. All rights reserved.
Contents
1.
2.
3.
Introduction ........................................................................................................................................................................................................................................................
Techniques for DNA-methylation analysis ...............................................................................................................................................................................................
2.1.
Techniques based on bisulfite treatment ....................................................................................................................................................................................
2.1.1.
Sequence-based analysis ..................................................................................................................................................................................................
2.1.2.
Melting temperature-based analysis ...........................................................................................................................................................................
2.1.3.
Interaction-based analysis ...............................................................................................................................................................................................
2.2.
Techniques based on biological recognition ..............................................................................................................................................................................
2.2.1.
Enzymatic digestion ..........................................................................................................................................................................................................
2.2.2.
Bioaffinity reaction ............................................................................................................................................................................................................
2.3.
Bisulfite-free and enzyme-free techniques ................................................................................................................................................................................
2.3.1.
Direct-oxidation-based analysis ....................................................................................................................................................................................
2.3.2.
Chemical-oxidation cleavage-based analysis ...........................................................................................................................................................
Conclusions and perspectives .......................................................................................................................................................................................................................
Acknowledgement ............................................................................................................................................................................................................................................
References ............................................................................................................................................................................................................................................................
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1. Introduction
Abbreviations: 5-caC, 5-Carboxylcytosine; 5-foC, 5-Formylcytosine; 5-hmC,
5-Hydroxymethylcytosine; 5-mC, 5-Methylcytosine; C, Cytosine; CE, Capillary electrophoresis; LC, Liquid chromatography; LOD, Limit of detection; MBD, Methylbinding domain; MTase, Methyl transferase.
* Corresponding author. Tel.: +86 20 84112901; Fax: +86 20 84112901.
E-mail address: daizong@mail.sysu.edu.cn (Z. Dai).
** Corresponding author.
E-mail address: ceszxy@mail.sysu.edu.cn (X.-Y. Zou).
http://dx.doi.org/10.1016/j.trac.2015.03.025
0165-9936/© 2015 Elsevier B.V. All rights reserved.
DNA methylation is a most important epigenetic modification
mode that plays key roles in gene-expression control [1]. DNA methylation exists in all living creatures and has several forms. In
prokaryotes, it usually occurs on cytosine (C) in CCA/TGG and GATC.
In eukaryotes, it occurs on purine, guanine, and mostly on C [2].
Under the catalysis of MTase, the fifth carbon of the C in dinucleotide 5′-CG-3′ can be selectively modified with a methyl group to form
5-mC. About 60–90% of 5′-CG-3′ dinucleotide in eukaryotes are methylated [3], and the unmethylated 5′-CG-3′ dinucleotide mostly
L. Zhang et al./Trends in Analytical Chemistry 72 (2015) 114–122
assembles in the structural gene promoter and the transcription start
point to form “CpG islands”.
The pattern of DNA methylation is inheritable [4] and changeable [5]. Normal expression of parental allele results in right gene
expression and abnormal DNA methylation in parental allele generally causes miscellaneous cancers [6], genetic diseases and ageing
abnormality [7]. Research for DNA methylation therefore has critical significance for medicine, life science and biochemistry.
In 2003, a world-wide human epigenome plan (HEP) was
launched, aiming to map the mutable sites of human DNA methylation and systematically study DNA methylation for tumor
formation, human epigenetics, embryonic development and gene
imprinting. In the HEP, the development of techniques for DNAmethylation analysis is fundamental and pivotal work, which is the
focus of attention.
In the past 30 years, the techniques for DNA-methylation analysis have been diligently developed in three groups classified
according to the purpose of investigation:
•
•
•
liquid chromatography/electrospray ionization mass spectrometry (LC-ESI-MS) [8], LC-MS/MS [9], and SssI methylase analysis
[10]; these techniques mainly focus on global methylation analysis, which aims to quantify the total amount of 5-mC in the
genome;
split β-lactamase sensor [11], methylation-specific multiplex
ligation-dependent probe amplification [12], and methylationspecific single-strand conformation analysis [13]; these techniques
are for gene-specific methylation analysis, which aims to evaluate specific methylation loci in the genome; and,
searching for new methylation loci, which tries to reveal potential DNA-methylation loci in the genome; the approaches include
inter-methylated sites amplification [14], methylation-sensitive
restriction fingerprinting [15], and methylation-sensitive arbitrarily primed polymerase chain reaction [16].
These techniques for DNA-methylation analysis were summarized in several reviews in recent years [17–23]. However, these
reviews commonly focus on specific aspects, such as research [17],
the technology of methylation-specific quantum-dot fluorescence
resonance-energy transfer (MS-qFRET) [18], MS [19] and biomarkers
[20], and applications to body fluids [21], colon cancer [22], and the
demethylation process [23].
To date, there have been few reviews on the research methodology of DNA-methylation analysis. In this review, we describe typical
approaches to DNA-methylation analysis. Furthermore, we assess perspectives of the methodology research of DNA-methylation analysis.
115
We envisage this review being helpful to researchers designing novel
methods of DNA-methylation analysis.
2. Techniques for DNA-methylation analysis
The direct approach to DNA-methylation analysis is by utilizing
LC or capillary electrophoresis (CE) to analyze the hydrolyzed DNA.
The LC method was first utilized by Kuo in 1980 [24], and was
further enhanced with PCR to be high-performance LC-PCR (HPLCPCR) [25]. With the application of MS {e.g., hydrophilic interaction
liquid chromatography–tandem mass spectrometry (HILIC-MS/
MS) [26]}, the selectivity and the sensitivity of this technology were
obviously improved. The detection range and the limit of detection (LOD) for 5-mC were 0.2–100 ng mL −1 and 45 pg mL −1 ,
respectively, and, for 5-hmC, analysis were 0.1–50 ng mL−1 and
57 pg mL−1, respectively.
With the latest reported technique using octyl-modified
quaternized cellulose in CE, the LODs for C and 5-mC were 1.1 μg mL−1
and 1.5 μg mL−1, respectively [27]. These technologies are not sensitive enough, and are merely utilized for global methylation analysis.
In comparison with the above direct approaches, numerous other
approaches are indirect methods that rely on specific recognition
of DNA-methylation loci, and are followed with amplification of recognition processes or isolation of the recognized strands based on
the changes in the properties of DNA strands. These methods commonly provide higher specificity and sensitivity for the analysis of
DNA methylation. As to the methylation-loci-recognition method,
the approach used most is by bisulfite conversion, and lots of techniques have been developed based on bisulfite treatment in
combination (e.g., with CE, melting temperature, fluorescence, or
electrochemistry).
However, in terms of the reaction efficiency and procedural difficulty, the crude bisulfite treatment is not perfect and some
biological technologies are used (e.g., restriction enzyme, mCpG antibody, or MBD). These methods still have the disadvantages of high
cost and limited application range, leading to continuing investigation of new methods for methylation-loci recognition (e.g., the
chemical-oxidation cleavage).
Table 1 lists the typical techniques based on different means of
recognition in the course of time, and we discuss the features and
the limitations of each method in detail in the following.
2.1. Techniques based on bisulfite treatment
Bisulfite treatment is a widely used, effective method for sorting
5-mC from unmethylated bases. During bisulfite treatment, the 5-mC
Table 1
The typical determination techniques of DNA methylation in various periods
Approaches
Classification
1990–2000
2001–2010
Bisulfite treatment
Sequence
Bisulfite sequencing
MS-PCR
Ms-SnuPE
MS-DGGE
RRBS
Melting Temperature
Interaction
Biological recognition
Enzymatic digestion
Bio-affinity reaction
Bisulfite-free enzyme-free
techniques
Electrochemical analysis
Chemical-cleavage-based
analysis
COBRA
Methylight
HPCE
MS-MCA
HRM
MSO
MS-DBA
RLGS
MS-RE-PCR/Southern
Online-monitoring
MBD
COMPARE-MS
CEIA
MWCNTs/Ch/GCE
C11H8O2
NaIO4/LiBr
2011–2014
E-msLDR
Label-free technique
CCP-FRET
Zinc finger protein
PPyox/MWCNTs/GCE
COEXPAR
Ref.
[28,29]
[30]
[31]
[32,33]
[34]
[35]
[36–38]
[39,40]
[41,42]
[43,44]
[45]
[46,47]
[48]
[49]
[50,51]
[52,53]
[54]
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L. Zhang et al./Trends in Analytical Chemistry 72 (2015) 114–122
Fig. 1. The main analysis approaches for 5-mC after bisulfite treatment.
in the gene is unchanged while C is specifically converted to uracil
(U), which will be further turned into thymine (T) after PCR amplification. As a result, the methylation information of the gene is
transferred to sequence information. The features of the gene, such
as melting temperature and specific recognition interaction, are
altered due to the change of sequence, which leads the differences
between these features becoming the key to DNA-methylation analysis (Fig. 1).
2.1.1. Sequence-based analysis
The straightforward idea for distinguishing 5-mC from other bases
is by gene sequencing. In 1992, Frommer proposed the bisulfitesequencing method to determine the 5-mC from individual strands
[28]. DNA was first treated by sodium bisulfite, and amplified with
PCR. By gene sequencing the resultant product, the status of 5-mC
was obtained. This technique is suitable for multiplex DNAmethylation determination, while the needs for massive cloning and
sequencing processes make sample preparation tedious and take
a long time.
In 1996, Herman developed an advanced sequencing-based technique, named methylation-specific PCR (MS-PCR) [30]. Different from
the previous bisulfite-sequencing technique, two specific promoters were pre-designed according to the methylated and
unmethylated sequences. After treatment by sodium bisulfite, the
target sequence was amplified by PCR with two specific promoters. If the target sequence was methylated, the amplified 5-mC was
contained in the PCR products. This technique avoids a complicated sequencing process. However, the sequence and the methylation
loci need to be known in advance due to the need to pre-design the
primer, which means that the technique can only perform a qualitative determination.
In order to conquer the limitations of MS-PCR, a technique named
methylation-sensitive single-nucleotide primer extension (MsSnuPE) was designed by Gonzalgo in 1997 [31]. After bisulfite
treatment, the DNA was amplified by PCR and separated by electrophoresis. The resultant products were further used as the template
for primer extension. The 3′-end of the extensional primer was designed before methylation loci. After polymerization with isotopes
labeled dCTP and dTTP by Taq, the methylated template led to the
tag of dCTP being at the end of primer, while the unmethylated template caused the dTTP to join to the primer. By electrophoretic
separation and radiation analysis of the terminal extended products, the ratio C/T was easily obtained, and gave the relative
proportions of methylated and unmethylated target sequences. The
technique is not only suitable for identification of the methylation
status of each gene site, but also capable of determination of the
L. Zhang et al./Trends in Analytical Chemistry 72 (2015) 114–122
methylation level. However, the tedious nature of primer design and
radioactive contamination inevitably remain as limitations.
With intensive study of sequencing techniques, next-generation
sequencing-based techniques were developed and applied as powerful analysis methods. In 2005, Alexander proposed a reduced
representation bisulfite-sequencing (RRBS) technique [55], which
utilized the restriction enzyme of Msp I to digest the genome to
enrich the CpG sites, and further performed the sequencing to obtain
the methylation information of each single base.
In 2010, the RRBS technique was further developed and applied
to the analysis of clinical samples, including mouse embryonic stem
cells and colon tumors. [29]. After optimizing the protocol by minimizing input DNA channel, adjusting size-selection of DNA fragments,
and maximizing unmethylated DNA channel, a pipeline was formed
and could analyze the methylation bioinformatics. This pipeline is
able to identify the imperceptible changes of methylated gene according to putative gene-regulatory potential, and further performs
a statistical test relating to differential DNA methylation. Compared with other sequencing approaches, this technique can screen
more samples with the ability to detect new epigenetic alterations, while the cost advantage is obvious.
2.1.2. Melting temperature-based analysis
Bisulfite sequencing provides a solid support for wide mapping
of the genome, but, regardless of bisulfite PCR sequencing, reduced
representation bisulfite sequencing or paired-end sequencing, the
multiple operation and the less reliable results caused by complicated bases and incomplete bisulfite conversion remain arduous
challenges [56,57]. The idea of sequencing-free determination therefore needs consideration, and the analysis for the unique melting
temperature of 5-mC sequence is typical.
In 1999, Aggerhoim developed a methylation-specific denaturing gradient gel electrophoresis (MS-DGGE) method for determining
the level of 5-mC [32]. Bisulfite-treated DNA sequences were treated
with DGGE, in which the denaturant solubility relating to the melting
temperature was increased from top to bottom. DNA strands changed
to be dendroid and stopped moving at different areas of the gel due
to the unique melting temperature of the methylated and
unmethylated DNA strands. Depending on the result of the separation, the 5-mC level was measured. The technique is economical
and can provide comprehensive analysis of DNA methylation.
However, the separation efficiency needs to be improved. Although the separation capability of MS-DGGE can be enhanced by
high-performance CE (HPCE) [33], the influence of the complicated bases to the targets remains.
In 2001, methylation-specific melting-curve analysis (MSMCA) was designed for the determination of 5-mC status [34]. After
bisulfite treatment, the DNA strands were labeled with a fluorescence dye. Depending on the measurement of the fluorescence
intensity during melting, the melting curves of DNA strands were
achieved, and their corresponding melting temperature were detected by Lightcycle processing. Because the melting temperature
is in positive proportion to the CG content in DNA sequence, the
melting temperature of the unmethylated DNA strand is lower than
that of the methylated DNA strand due to the conversion of CG to
UG during bisulfite treatment. Therefore, the methylated and
unmethylated DNA strands were discriminated.
However, using this technique, it is difficult to detect the methylation status of each single base precisely. The limitation was
overcome by the enhanced approach of high-resolution melting
(HRM) analysis, which was proposed by Carlos to fulfill direct detection of 5-mC in 2010 [35]. Different from MS-MCA, HRM utilized
a saturable fluorescent dye to label the DNA sample. An obvious
change of fluorescence signal was obtained during the melting due
to the occupation of each target base site by the dyes. With high
117
resolution, the methylation status of each single base was precisely distinguished.
2.1.3. Interaction-based analysis
Besides melting-temperature analysis, some specific interactions can be considered as another way for economical, efficient
recognition of target sequence. Restriction enzymes and oligonucleotide (oligo) probes are widely used in the interaction, which
becomes the core of the approach.
In 1997, combined bisulfite-restriction analysis (COBRA) was first
proposed by Xiong and Peter for determination of the status of 5-mC
[36]. After bisulfite treatment and PCR amplification, the DNA sample
was added with the restriction enzyme of BstU I, which recognized and digested the methylated sequence of 5′-CGCG-3′. The
resultant products were analyzed by electrophore to determine the
5-mC status. The technique can easily provide accurate results, but
the application range is restricted due to the sequence-dependence
of the restriction enzyme.
At the beginning of the twenty-first century, technologies of enzymatic digestion and oligo-probe hybridization were joined together
for 5-mC analysis. The typical technique is Methylight [39]. A specific oligo probe, Tagman, was designed and labeled with fluorophore
and quencher on its 5′ and 3′-ends, respectively. After bisulfite treatment, DNA sample was incubated with the probe and analyzed by
real-time PCR. The duplex strands were cut by excision enzyme at
5-mC, and the fluorophore at the 5′-end was released. From the fluorescence signal produced, the level of 5-mC was quantified. In order
to improve the throughput of analysis further, an enhanced technique, termed methylation-specific oligo (MSO) micro-array, was
constructed by using multiple oligo probes for methylated and
unmethylated DNA [37]. However, the applications of excision
enzyme and real-time PCR led to an uneconomical, difficult
procedure.
In 2013, our group proposed a PCR-free method, termed electrochemical methylation-specific ligation-detection reaction (EmsLDR), to quantify multiplex gene-specific DNA methylation [38].
Two pairs of oligo probes were designed for two methylation loci.
Reporting probes were labeled with electrochemical quantum dots
(QDs) of PbS and CdS, respectively, and capture probes were immobilized on magnetic beads as the isolation vehicle. After bisulfite
treatment, the target gene was selectively hybridized on the isolation vehicle with the QD-labeled reporting probes. Under catalysis
of E. coli DNA ligase, the reporting probes were specifically interlinked with capture probes at methylated loci. From the types and
the amount of linked QDs on the isolation vehicle, the pattern and
the levels of the two gene-specific DNA-methylation loci were simultaneously evaluated. The main advantage of this technique is
avoiding the PCR process and restriction-enzyme reaction, but the
preparation of multiple probes and the labeling procedure are
complex.
Besides oligo probes, some proteins with bioaffinity to methylation loci were also utilized to analyze 5-mC status, such as
methylation-sensitive dot-blot assay (MS-DBA) [40]. After bisulfite treatment and PCR amplification, the DNA sample was incubated
with DIG-labeled methylation-sensitive dinucleotide probe and then
with luciferin-labeled DIG antibody. From the signal of luciferin, the
methylation status of the target was determined. The technique has
the capacity for multiplex 5-mC analysis, while the difficult bisulfite treatment and labeling procedure remain in the operational
procedure.
2.2. Techniques based on biological recognition
Although bisulfite treatment has been widely utilized to distinguish 5-mC from other C, it has issues of incomplete conversion,
false-positive results, difficult operation, and being time consuming.
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Fig. 2. The main biological recognition approaches for 5-mC and the determination ways for the recognized targets.
In comparison, biological identification approaches, such as enzymatic digestion or bioaffinity reaction, can rapidly recognize
methylation loci with high specificity under mild reaction conditions, so the 5-mC analysis methods based on biological recognition
have become a new focus of attention (Fig. 2).
2.2.1. Enzymatic digestion
Since the beginning of the twenty-first century, techniques associated with enzymes have been widely applied to studies of DNA
methylation. The typical method is restriction-landmark genomic
scanning (RLGS) [41]. DNA sample was digested by the Not I restriction enzyme, and resulted in containing the methylated loci.
The sequences were then labeled with 32P-dCTP and 32P-dGTP and
digested by methylation-insensitivity restriction enzyme (EcoR V),
and, after preliminary separation by performing one-dimensional
electrophoresis, the product was further treated with another highfrequency methylation-insensitivity restriction enzyme (Hinf I) and
subjected to two-dimensional electrophoresis. After two rounds of
screening, the methylation status of multiple CpG sites was investigated. However, operation of the technique is complicated, and
the result is uncertain.
In 2004, an advanced enzyme-digestion technique, named
methylation-sensitive restriction endonuclease-PCR/Southern (MSRE-PCR/Southern), was developed [43]. DNA samples were treated
with Hpa II and Msp I restriction enzymes. Both Hpa II and Msp I
enzymes can specifically recognize the 5′-CCGG-3′ sequence. Hpa
II and Msp I cannot digest the unmethylated C in 5′-mCCGG-3′, while
Msp I can cleave the methylated C in 5′-CmCGG-3′. DNA samples were
digested with Hpa II and Msp I enzymes, and analyzed by PCR or
Southern blot. The status of DNA methylation at multiple methylation loci was obtained. The technique is simple and economical, but
it is not suitable for complex gene samples.
In 2007, methylation-sensitive restriction endonuclease was utilized to monitor the activity of MTase on-line and evaluate the
methylation status [45]. A molecular beacon labeled with a pair of
fluorophore and quencher was used as oligo probe. The probe initially
showed a low fluorescence signal in its hairpin form. Under the catalysis of Dam MTase, 5-mC fragments were generated in the probes.
The addition of Dpn I endonuclease then specifically cleaved the
strand at the methylated site, resulting in an increase in the
fluorophore signal due to the separation of fluorophore and quencher.
The activity of MTase and the DNA-methylation status were therefore monitored from the obvious fluorescence signal. Nevertheless,
the procedure and the cost of fluorescent labeling are drawbacks
of this technique.
In 2012, a label-free, PCR-free electrochemical method was proposed by our group for gene-specific methylation analysis [42]. Oligo
probes were covalently attached on a 3-mercaptopropionic acid selfassembled monolayer-modified gold electrode. The amount of the
oligo probe was shown by the peak current (I1) of the electrochemical indicator of methylene blue (MB) before hybridization. After the
hybridization of sample strands on the electrode with probes, the
peak current of MB reduced to I2 due to wrapping of guanine in the
hybridized DNA strands. Under the catalysis of Hpa II endonuclease, unmethylated DNA duplex strands were cleaved at the genespecific methylation locus, resulting in a decrease in the peak current
to I3. Thus, the degree of 5-mC was evaluated according to the equation:
Methylation Degree = (1 − ( I2 − I3 ) ( I1 − I2 )) × 100%
The technique showed satisfactory selectivity and sensitivity with
an easy operational procedure. However, the limitation for multipletarget determination remained.
In 2013, cationic conjugated polymer-based FRET (CCP-FRET) was
designed for multiple DNA determination [44]. DNA samples were
digested by methylation-sensitive restriction endonuclease (Hpa II)
and then subjected to a two-round PCR with fluorescein-labeled
dNTPs (Fl-dNTP) and dNTP/Fl-dNTP, respectively. After cleavage of
the impurities by shrimp alkaline phosphate (SAP), CCP was added.
The fluorescence signal changed because the fluorescence resonance energy transfers from CCP to the fluorescein tag, indicating
L. Zhang et al./Trends in Analytical Chemistry 72 (2015) 114–122
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Fig. 3. Two types of bisulfite-free and enzyme-free analysis approaches. One is measuring the produced signals of the oxidation interaction, and the other is measuring the
target fragments cleaved by performing the chemical interaction.
the level of 5-mC. This technique was applied in cancer detection
with the sensitivity threshold of 85.7%, but it needs 20 h to complete the determination.
2.2.2. Bioaffinity reaction
Some biomolecules, such as MBD, anti-5-methylcytosine IgG1 antibody, zinc finger (ZF) protein, show inherent capability of
methylation-loci specificity and can be directly used for DNAmethylation analysis [46–49,58]. In 2004, Shiraishi proposed an MBDbased method for screening and identifying 5-mC in the genome
[46]. The exposed polypeptide functional area of MBD can strongly
combine with 5-mC fragments, and the combination ability is in positive proportion to the level of the 5-mC. In combination with column
chromatography, the 5-mC was therefore directly separated from
other strands. However, the non-specific binding of co-existing DNA
strands may influence the accuracy of analysis.
In 2006, an enhanced technique was proposed by combining
methylated-DNA precipitation with methylation-sensitive restriction enzyme (COMPARE-MS). DNA sample was digested with
restriction enzymes, and the 5-mC fragments were isolated by the
MBD column and amplified by PCR amplification [48]. The technique took advantage of the sensitivity and the specificity of MBD,
and effectively avoided the false-positive result that commonly exists
in MBD-based methods. However, the sequence dependence of the
restriction endonuclease limits the method in the use of wide range
of DNA sequences.
Besides MBD, anti-5-methylcytosine IgG1 antibody was utilized for the construction of a CE immunoassay (CEIA) in 2009 [49].
The 5-mC fragments were recognized by anti-5-methylcytosine IgG1
antibody, and then combined with the antigen labeled by a monovalent fluorescein (i.e., Fc-specific anti-IgG1 Fab fragment). The
immune complex of the 5-mC formed was then separated and determined by CE and on-line laser-induced fluorescence polarization,
respectively. The LOD of 5-mC in this economical, handy technique was 0.3 nM. The method can avoid tedious bisulfite
pretreatment, enzyme digestion, and PCR-amplification processes
of DNA-methylation determination, and simplify the analytical
operation.
ZF protein, as another popular DNA-binding protein in mammals
[59], was applied to methylation analysis in 2012 [47]. DNA strands
containing the target region were first isolated from the sample by
sonication. The methylated strands were then captured by MBD and
amplified by PCR. After that, luciferase-fused ZF protein was used
to recognize and to bind to the methylation locus. From the determination of luciferin, 5-mC was sensitively quantified. The technique
had good performance with high accuracy, sensitivity, and potential for automation.
2.3. Bisulfite-free and enzyme-free techniques
Although bisulfite-conversion and biological methods show high
specificity for recognition of DNA-methylation loci, the issues of reaction efficiency, analytical cost and operational complexity remain
of concern in 5-mC analysis. A bisulfite-free, enzyme-free method
for DNA methylation would conquer these limitations and possess
numerous benefits, such as speed, convenience and low cost, which
would be helpful for current techniques (Fig. 3).
2.3.1. Direct-oxidation-based analysis
The C5-C6 double bonds of thymine and 5-mC are oxidized by
osmium tetroxide [50,51,60,61], so there is a good potential way to
utilize the direct electrochemical oxidation of pyrimidine for the
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analysis of DNA methylation. In 2010, an electrochemical method
was proposed for global DNA-methylation analysis [50]. A choline
chloride monolayer supported multiwalled carbon nanotubes
(MWCNTs) film-modified glassy-carbon electrode (MWCNTs/Ch/
GCE) was fabricated. MWCNTs provide remarkable electrocatalytic
activities for DNA bases. All purine and pyrimidine bases of guanine,
thymine, adenine, C and 5-mC were identified by their unique oxidation signals.
In the following years, an enhancement technique was designed to improve the determination capacity for mixed bases by
using over-oxidized polypyrrole directed MWCNTs/GCE (PPyox/
MWCNTs/GCE) [51]. The technique possesses preeminent specificity
and accuracy, and can be applied for the rapid determination of
mixed bases without enzyme, probe or bisulfite treatment.
2.3.2. Chemical-oxidation cleavage-based analysis
Recently, several new chemical methods based on chemicaloxidation cleavage were proposed for discrimination of 5-mC. With
the inspiration of the distinctive capability of OsO4 for 5-mC from
C, Yamata et al. developed a method employing 2-methyl-1,4naphthoquinone-chromophore to identify 5-mC through
photosensitized oxidation [52]. Since then, an advanced method
using NaIO4/LiBr was reported [54]. The C5-C6 double bond of 5-mC
in DNA was selectively oxidized by NaIO4/LiBr, and was subsequently cut by hot piperidine treatment. The chemically-cleaved
DNAs were analyzed by denaturing polyacrylamide gel electrophoresis (PAGE), and the DNA-methylation status was easily obtained.
The chemical-cleavage method shows high efficiency for
methylation-loci recognition, but the lack of sequence selectivity
and the unsatisfactory sensitivity hinder its application in genespecific methylation analysis. With respect to these issues, we
recently developed a technique termed chemical-oxidation cleavage triggered exponential amplification reaction (COEXPAR). Target
DNA was first treated with NaIO4/LiBr solution. The C5-C6 double
bond of 5-mC was selectively epoxidized and brominated to be susceptible to cleavage. The following hot piperidine treatment cleaved
the methylated target DNA at 5-mC to produce a short primer for
the initiation of exponential amplification reaction (EXPAR), resulting in an amplified fluorescence signal related to the quantity of
5-mC. This technique showed not only good sequence selectivity,
but also high amplification efficiency and rapid amplification kinetics. The COEXPAR method successfully analyzed gene-specific
methylation in real time, and was ultrasensitive with an LOD down
to 411 aM [53].
3. Conclusions and perspectives
With the advances of HEP, technologies for DNA-methylation
analysis are continually progressing, and remain key in mapping the
mutable sites of human DNA methylation and systematically studying DNA methylation. The current methods for DNA-methylation
analysis can fulfill the determination of 5-mC and the relevant oxidation products in an efficient way, while the deficiencies on target
recognition remain severe challenges. Meanwhile, the analysis of
epigenetic modifications in the demethylation process is becoming a focus of attention, which brings new aspects of research.
Current ways of recognizing 5-mC mainly include biological approaches, such as probe hybridization, enzyme digestion and protein
combination, and chemical approaches, such as bisulfite treatment and chemical oxidation. Probe-recognition techniques, such
as padlock probe [62], linear padlock probe [63] and molecular inversion probe [64], have been widely applied in DNA-methylation
analysis due to their admirable specificity and flexible formation.
By artful capture of target fragments, these techniques can provide
a quick, auto-circling and cost-effective ways of discrimination.
However, they lack the capacity for multiple-target recognition.
The enzyme-digestion and protein-combination technologies
possess high sensitivity and specificity for distinguishing targets
[65,66]; while they are similar to probe-based techniques, the complicated targets normally cause uncertain results. In comparison,
chemical-recognition methods have the advantage of highthroughput sample discrimination, but skillful operation is needed.
Very recently, a novel approach based on nanopore became the
focus of attention. Under electric field, the 5-mC base was forced
through a membrane containing a synthetic nanopore, which allowed
only one molecule to cross at a time. From the change of voltage
threshold, the amount of the pervasive 5-mC was determined [67].
The nanopore-based technique was further developed in the following years. For example, an oligonucleotide modified α-hemolysin
(αHL) protein nanopore was equipped within a cyclodextrin adapter.
This synthetic complex possesses three recognition points with the
ability to identify individual unmodified nucleobases [68]. Furthermore, combined with the enzymes of phi29 DNAP and M2MspA,
the analytical performance of the synthetic nanopore device was
enhanced to distinguish the variants of 5-mC, such as 5-hmC, 5-foC
and 5-caC, and the accuracy range for the variants was 91.6–98.3%
[69]. The nanopore technology provides unique inspiration in the
ways of recognizing methylated genes.
Recent reports indicated that active DNA demethylation may be
achieved through multistep oxidation of 5-mC with the generation of intermediates of 5-hmC, 5-foC, and 5-caC. This novel means
of identification indicates that DNA modifications possess potential regulatory roles [70]. Different from 5-mC, 5-hmC has an
abundance within the genome 10–100-fold lower than that of 5-mC,
and the abundances of 5-foC and 5-caC are about 40–1000-fold
higher than that of 5-hmC [70–72]. These C modifications are a hot
spot, and the analytical methods have been developed significantly, and mainly involve the application of LC, CE, bioaffinity or
chemical derivatization, such as recombinant β-glucosyltransferase
analysis [73], selective chemical labeling [74], combined glycosylation
restriction analysis [75], and TAB-array [76].
Besides modification analysis, the investigation of enzymology
is becoming a new focus of research. Some 10–11 translocation
enzymes (TET1-3) are well-known oxidases for the conversion from
5-mC to 5-hmC, 5-foC or 5-caC [77,78], which is a potential starting point for the analysis of methylated DNA derivatives.
Acknowledgement
This work was supported by the National Natural Science
Foundation of China (21175158, 21375154, 21422510, and
81171666), the Natural Science Foundation of Guangdong Province (S2013010012135), and the Ph.D. Programs Foundation of the
Ministry of Education of the People’s Republic of China
(20110171110014).
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ID
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Title
DevelopmentoftechniquesforDNA-methylationanalysis
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