Droplet Digital PCR

DNA Methylation Detection™
Using Droplet Digital PCR

Hamutal Bonen, PhD; Jeremiah McDole, PhD; Eddy van Collenburg
Bio-Rad Laboratories, Inc., Digital Biology Center, 5731 W Las Positas Blvd, Pleasanton, CA 94588

Abstract

The year 2023 marks 12 years since the advent of Droplet Digital PCR (ddPCR™). In that time, ddPCR
technology has revolutionized the absolute quantification of nucleic acids. With a host of technical advancesand enhanced automation in recent years, Droplet Digital PCR is moving from a specialized researchtechnique to a robust tool in clinical and translation research applications. This white paper reviews theuse of Droplet Digital PCR for detection of DNA methylation. We review ddPCR technology, compare
different workflow strategies, and explain critical parameters for optimal assay design to get unprecedented
sensitivity, linearity, and robustness when detecting DNA methylation by Droplet Digital PCR.
Introduction
DNA methylation plays a critical role in many normal developmental
and sustaining physiological processes. However, aberrant
methylation can lead to a variety of disease states, including
several types of cancer. For instance, global hypomethylation cancontribute to the activation of oncogenes while hypermethylation
within tumor suppressor genes can result in silencing. Mounting
evidence demonstrates the utility of analyzing methylation status
as an indicator of early-stage cancer development and as amethod for monitoring tumor response to therapy. Detecting
methylation changes in cell-free DNA (cfDNA) is especiallyattractive given the noninvasive nature of blood draws.
Unfortunately, sensitive, robust, and cost-effective methods toperform methylation analysis on cfDNA samples remain an unmet
need. Here we demonstrate how common methylation detection
methods combine with Droplet Digital PCR to enhance analysis ofthese precious samples.
DNA Digestion Using Methylation-Sensitive Restriction
Enzymes (MSRE)
By far, the fastest and easiest method of DNA methylation
quantification incorporates MSRE digestion. Further, this method does
not have the drawbacks of bisulfite conversion, such as extensive
DNA degradation and high starting material input requirements.

Bulletin 3585

As shown in the schematic below, methylation-sensitive restriction
enzymes do not cleave restriction sites in the presence of amethylated cytosine, leading to PCR amplification (Figure 1), whilecleavage of an unmethylated site inhibits amplification.

Unmethylated
Restriction Site

No
Amplification

Cleavage
MSRE

PCR

M

M

Methylated
Restriction Site

No Cleavage

Amplification

Fig. 1. Schematic overview of methylation-sensitive restriction enzyme
digestion. M, methyl; MSRE, methylation-sensitive restriction enzyme.

There are a variety of commercially available MSREs available(Table 1). Specific primers can be designed to amplify a specificsequence of interest that contains an MSRE. See the Primer
Design section for recommendations on how to design primers.

DNA Methylation Detection Using Droplet Digital PCR

Samples are prepared as follows (incubations can be done in athermal cycler such as the T100 Thermal Cycler):

Table 1. Overview of MSREs and restriction site sequences.
Restriction Enzyme (all 4-base cutters)

Restriction Site

AccII (BstUI, Bsh1236I)

CG↓CG
GC↑GC

AciI (SsiI)

C↓CGC
GGC↑G

HpaII (HapII)

C↓CGG
GGC↑C

HhaI (CfoI)

GCG↓C
C↑GCG

■

■

 SRE+ samples — 50–100 ng of DNA is incubated for 1 hr at
M
60°C with 2–10 U of BstUI and 0.5 µl 10x CutSmart Buffer (NewEngland Biolabs, Inc., discontinued; rCutSmart Buffer [#B6004S]
is a suitable alternative) in a total volume of 5.0 μl.
 SRE– samples — 50–100 ng of DNA is incubated for 1 hr at
M
60°C with 0.5 μl 10x CutSmart Buffer in a total volume of 5.0 μl.

In a 22 µl experiment, 1–100 ng of incubated DNA samples can beanalyzed using 11 µl ddPCR Supermix for Probes (No dUTP)(Bio-Rad Laboratories, Inc., catalog #1863024) and primers andprobes in a final concentration of 900 and 250 nM, respectively.

To detect the methylation status at a specific locus, the sample
is digested by a methylation-sensitive restriction enzyme andanalyzed with Droplet Digital PCR (Figure 2). When using probebased detection methods, multiplexing several methylation sites
or targets is possible. For best results, a duplex reaction with areference target (no restriction sites) is advised (e.g., RPP30).
The reference assay is used to normalize and correct for small
input differences caused by pipetting or samples with unstablecopy number.

PCR mixtures are partitioned into ~20,000 droplets using theAutomated Droplet Generator (Bio-Rad, #1864101). Subsequent
PCR is performed in a thermal cycler using the the protocol
outlined in Table 2.
Table 2. Thermal cycling protocol. Ramp rate set to 2°C/sec for all steps.

Methylation percentages are calculated with high precision via
Droplet Digital PCR by measuring DNA concentrations in samples
with (MSRE+) and without (MSRE–) enzyme. The percentage ofmethylation, or methylation fraction, is calculated using the precise
ddPCR concentrations measured for samples either digested(MSRE+) or undigested (MSRE–), as follows:

Cycling Step

Temperature, °C

Time

Number of Cycles

Enzyme activation95

10 min1

Denaturation95

30 secAnnealing/extension

55

1 min

Enzyme deactivation98

10 minHold

12<48 hr40
1Droplet reading can be performed on a QX200™ or QX600™
Droplet Reader. All the data shown in this white paper weregenerated using a QX200 Droplet Reader.

MSRE+ ratio = target concentration/reference concentration
MSRE– ratio = target concentration/reference concentration% methylation = MSRE+ ratio/MSRE– ratio
Note that copy number results are available from the undigested(MSRE–) samples and can be used to detect differences insamples (assuming the use of a stable reference gene).

MSRE+ Samples
+

+

+

+

10

10

Follow provider instructions

+

+

+

MSRE– Samples

ddPCR master mix
containing assay(s)and sample

Droplet generation

PCR amplificationby thermal cycling

Droplet reading on a
QX200 or QX600
Droplet Reader

Fig. 2. Typical ddPCR MSRE workflow. Sample is split. One aliquot is digested with a restriction enzyme and the other without restriction enzyme. Both aliquots are run as
individual samples using a standard ddPCR setup. MSRE, methylation-sensitive restriction enzyme.

© 2023 Bio-Rad Laboratories, Inc.

2

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DNA Methylation Detection Using Droplet Digital PCR

Examples of Assay Performance
In this section, we highlight assay performance using a few
examples (Figures 3–5).

SEPT9 CGI3 Promotor (FAM) and RPP30 (HEX)
FAM
9,000

FAM

8,000
7,000

6,000

6,000
Amplitude

5,000

Amplitude

4,000
3,000

5,000
4,000
3,000
2,000

2,000

1,000
0

1,000

–1,0000HEX–1,000

6,000

HEX

5,000

5,000

4,000

Amplitude

Amplitude

4,000
3,000

3,000
2,000

2,000

1,000
0

1,000
9,000

0

8,000
7,000
FAM amplitude

6,000

FAM amplitude

5,000
4,000
3,000
2,000

6,000
5,000
4,000
3,000
2,000
1,000

1,000

0
–1,00000–1,000
1,000

2,000
3,000
HEX amplitude

4,000

5,000
Concentration,
copies/μl

Concentration, copies/μl

0

300
250

252

200
150

183

179

186

272

249

175

100
50

1,000

400
350
300
250
200
150
100
50
0

2,000
3,000
4,000
HEX amplitude

350

6,000

271

272

335
217

Methylated MSRE+

2.39

Methylated MSRE–

Methylated MSRE+

Unmethylated
MSRE–

Fig. 3. PTGER4 promotor (FAM) and RPP30 (HEX). One-dimensional plots show
the positive and negative droplet clusters from two targets run in duplex, PTGER4(FAM, AssayID dHsaEXD82842910) and RPP30 (HEX, AssayID dHsaCP2500350).
Two-dimensional plot shows the four clusters from the same reaction. HpaII is used
for digestion. Concentrations (copies/μl) are shown in the bottom graph and used
for calculating the percentage of methylation from each sample (see calculations).
MSRE, methylation-sensitive restriction enzyme.

Table 4. SEPT9 promoter methylation calculations.

Table 3. PTGER4 promoter methylation calculations.
MSRE+ Ratio

175/185 = 0.946

2.39/249 = 0.0096

MSRE– Ratio

179/183 = 0.978272/252 = 1.079

Methylation, %0.946/0.978 = 97%

0.0096/1.079 = 0.9%

© 2023 Bio-Rad Laboratories, Inc.

Unmethylated
MSRE+

The percentage of SEPT9 promoter methylation was calculated asshown in Table 4.

PTGER4 Promoter (FAM) and RPP30 (HEX)The percentage of PTGER4 promoter methylation was calculated
as shown in Table 3.
UnmethylatedUnmethylated
MSRE–

Fig. 4. SEPT9 CGI3 promotor (FAM) and RPP30 (HEX). One-dimensional plotsshow the positive and negative droplet clusters from two targets run in duplex,
SEPT9 CGI3 (FAM, AssayID dHsaEXD18126781) and RPP30 (HEX, AssayID
dHsaCP2500350). Two-dimensional plot shows the four clusters from the same
reaction. BstUI is used for digestion. Concentrations (copies/µl) are shown in thebottom graph and used for calculating the percentage of methylation from eachsample (see calculations). MSRE, methylation-sensitive restriction enzyme.

Unmethylated
MSRE+

Methylated

266

208

2.33

Methylated MSRE–

0

5,000

3

MethylatedUnmethylated

MSRE+ Ratio

335/208 = 1.611

2.33/266 = 0.0088

MSRE– Ratio

350/217 = 1.613

271/272 = 0.996

Methylation, %1.611/1.613 = 100%

0.0088/0.996 = 0.9%

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DNA Methylation Detection Using Droplet Digital PCR

Probe Mix Triplex WIF1 (FAM), NPY (HEX), and RPP30 (0.4x FAM, 0.25x HEX)WIF1 and NPY methylation was calculated as shown in Table 5.

FAM

Table 5. WIF1 and NPY methylation calculations.

18,00016,000

Amplitude

12,00010,0008,000

MSRE+ Ratio

0.345/56 = 0.0062

0.0559/53.7 = 0.00104

MSRE– Ratio

53.7/57.9 = 0.927

53.7/57.9 = 0.927

Methylation, %0.0062/0.927 = 0.7%

0.00104/0.927 = 0.1%

6,000
4,000

MSRE+ Ratio

5.48/56 = 0.0979

5.16/53.7 = 0.0961

2,000

MSRE– Ratio

58.3/57.9 = 1.007

58.3/57.9 = 1.007

Methylation, %0.0979/1.007 = 9.7%

0.0961/1.007 = 9.5%

HEX

ddPCR Assay Design
Since analysis is based on restriction enzyme cleavage, theamplicon sequence must include cleavage sites. This approachprovides high specificity, however only specific restriction sites canbe analyzed, which can be a limitation. To reliably measure DNA
methylation, we recommend including at least two but not morethan four restriction sites in the amplicon. If a single MSRE cannot
cut at least two sites in an amplicon, combining multiple MSREs
that cut within the amplicon is a good alternative. For an extended
overview on how to design specific ddPCR assays, please refer tothe Assay Design for Droplet Digital PCR section in bulletin 6407.

6,000
5,000
Amplitude

ddPCR Master Mix Digestion

NPY

0

4,000
3,000
2,000
1,000
0
18,00016,00014,000

FAM amplitude

Overnight Digestion
WIF1

14,000Consider the following when designing primers:

12,00010,000

■

8,000
6,000

■

4,000
2,000
■

0
0

1,000

2,000

3,000

4,000

5,000

6,000

Concentration, copies/μl

HEX amplitude
70
60
50
40
30
20
10
0

57.9

53.7

58.3

57.9
55.4
52.1

■

57.5

56

53.7

56.7
■

5.48
0.345

Unmethylated MSRE–

5.31
0.321

Unmethylated MSRE+(Buffer)

5.16
0.05595.01
0.217

Unmethylated MSRE+(Sample)■Fig. 5. Probe Mix Triplex WIF1 (FAM), NPY (FAM), and RPP30 (0.4x FAM, 0.25x
HEX). One-dimensional plots show the positive and negative droplet clusters fromthree targets run in triplex. WIF1 (FAM, AssayID dHsaEXD31159278), NPY (FAM,
AssayID dHsaEXD18238370), and RPP30 (0.4x FAM, AssayID dHsaCP2500313,
0.25x HEX, AssayID dHsaCP2500350). Two-dimensional plot shows up to eightclusters from the same reaction. A mixture of AciI and HhaI was used for digestion.
Two replicate reactions of an unmethylated control sample were used to compare
the digestion in sample (buffer: digestion overnight) with the digestion in master mix(sample: 45 min at room temperature). Concentrations (copies/μl) are shown in thebottom graph and used for calculating the percentage of methylation from eachsample (see calculations). MSRE, methylation-sensitive restriction enzyme.

Check for restriction sites within the area of your target sequence ook for restriction enzymes that have 2 or more restriction sites
Lclose together
 esign primers outside these restriction sites to produce anDamplicon with 2 or more restriction sites
 restriction site can be included in the primers but only inAthe last 4 nucleotides of the 3' end of each of the primers.
Restriction sites outside the primers are preferred hort amplicons (<100 nucleotides) enable fragmented sample
S
analysis (e.g., formalin-fixed paraffin-embedded [FFPE] orcirculating tumor DNA [ctDNA]) ombining different restriction enzymes is possible and used toCperform double digestions in the same reaction

Example Assay Design

Figure 6 shows an example assay design for the transcription
factor one cut homeobox 2 (ONECUT2).
Amplicon: 64 bp, location hg19|chr18:55103715-55103778:+
Sequence: AAATGCTCAGCCCCAACTTCGACGCGCACCACACTGCCATGCTGACCCGCGGTGAGCAACACC
Fig. 6. Example assay design for the transcription factor ONECUT2 (BstUI
restriction sites). Bio-Rad PrimePCR AssayID: dHsaEXD31605634. BstUI sites arebolded, primer sites are underlined, and the probe site is highlighted yellow. Chr,
chromosome; hg, human genome.

© 2023 Bio-Rad Laboratories, Inc.

4

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DNA Methylation Detection Using Droplet Digital PCR

Cytosine Deamination
While MSRE digestion protocols are rapid and reliable, sodium
bisulfite conversion is the current gold standard of methylation
analysis. In this method, DNA is denatured and treated with sodium
bisulfite. This process deaminates unmethylated cytosine in DNA touracil and leaves methylated cytosines unchanged. Amplifying thetreated DNA with PCR converts uracils further to thymines while
the methylated cytosines remain cytosines. An example of cytosine
deamination is shown in Figure 7.

Primer Design

Although bisulfite treatment is relatively fast and straightforward,
it requires elevated temperatures and high pH that candamage DNA, resulting in fragmentation and depyrimidination.
Unmethylated cytosines are especially sensitive to degradation
with this method, resulting in challenges amplifying GC-rich areas.

■

There are two main design strategies available to specifically
amplify converted (unmethylated) and unconverted (methylated)sequences with Droplet Digital PCR: methylation-specific primers
and methylation-independent primers (Figure 8).
Primer design considerations for methylation-specific primers:■■

Include as many CpG sites as possible, especially at the 3' endof the primer
 onsider the same CpG sites in the primer sequence forCmethylated DNA and unmethylated DNA
Maximize specificity by using a detection probe including CpG sites

Primer design considerations for methylation-independent primers:
No CpG sites within the primer sequence
Use an adequate number of C (no CpG)
■ Spanning a maximum number of CpG sites in the amplicon■ Maximize specificity by using a detection probe including CpG sites■

A less damaging method for cytosine deamination was recently
developed by New England Biolabs, Inc. (NEB). In this method,
APOBEC enzyme is used to convert cytosine to uracil, resulting in
far less DNA degradation. However, APOBEC can also deaminate
5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). To
protect the methylated cytosine from deamination, NEB uses TET2
enzyme and Oxidation Enhancer (#E7129) to modify 5mC and5hmC to forms that are not substrates for APOBEC.

■MSP Design Schematic
Two Different Primer Pairs

When deciding which method to utilize, the required starting
material should be considered. Sodium bisulfite protocols
typically require between 0.1 and 2 μg of DNA (depending on the
manufacturer) while the enzymatic approach can be performed
with much less.

Unmethylated
CpG Sites

MethylatedSince both methods rely on the conversion of unmethylated DNA
to thymine, primers and probe designs are the same for both.
Template

A: 5'–GACCGTTCCAGGTCCAGCAGTGCGCT –3'
B: 3'–CTGGCAAGGTCCAGGTCGTCACGCGA –5'

Cytosine
Deamination

A: 5'–GATCGTTTTAGGTTTAGTAGTGCGTT –3'
B: 3'–TTGGCAAGGTTTAGGTTGTTATGCGA –5'

MIP Design Schematic
Same Primer Pairs

Detection Probes to Multiplex Targets/Loci

Fig. 7. Example of cytosine deamination on template DNA strand. Underline
denotes methylated cytosine. Note that sense and antisense strands are no longer
complementary after deamination.
Fig. 8. Comparison of methylation-specific and methylation-independent
design strategies. CpG, 5'-cytosine-phosphate-guanine-3'.

© 2023 Bio-Rad Laboratories, Inc.

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DNA Methylation Detection Using Droplet Digital PCR

Summary
Methylation-sensitive primers have the advantage of detecting
methylation levels at the primer-binding region but have thedisadvantage of needing two different primer sets.

Example Performance (Rat SNRPN )Figure 9 shows examples of the performance of methylationspecific and methylation-independent primers.
Ch1+Ch2+:2284, Ch1+Ch2–:14314, Ch1–Ch2+:59623, Ch1–Ch2–:189385

Methylation-insensitive primers have the advantage of a single
primer set, which should compensate for PCR bias and canbe combined with high resolution melt analysis, resulting inmethylation information at the regional level (amplicon).

12,000Channel 1 amplitude

10,0008,000

Neither method gives information on individual CpG resolution levels.
Conclusions
Droplet Digital PCR has been utilized in many new genetic analysis
applications. Adding methylation-based detection tools to its
already large portfolio of applications highlights the continuously
growing utility of Droplet Digital PCR.

6,000

4,000

2,000

While many conventional methods are available for cytosine
deamination–based methylation analysis (methylation-specific
PCR, high resolution melt analysis, pyrosequencing, etc.), several
publications have demonstrated the added value that Droplet
Digital PCR provides (van Wesenbeeck et al. 2018, Cho et al. 2020,
Hagelans et al. 2017, Lehmann-Werman et al. 2018, Larsen et al.
2018, Jensen et al. 2019).

0
0

1,000

2,000

3,000
4,000
Channel 2 amplitude

5,000

Methylated sense forward		
Methylated sense reverse		
Methylated probe		

TATTGTAGTTAGAGAGTGATATGGATTCTAAACTTACCGCAATAACTCAAAT
FAM-ACTACTCCCTACGCATACGTCCAAA

Unmethylated sense forward
Unmethylated sense reverse
Unmethylated probe		

TTGTAGTTAGAGAGTGATATGGATTACTAAACTTACCACAATAACTCAAATHEX-ATACTACTCCCTACACATACATCCAAAACA

6,000

Methylation detection using MSRE-based digestion in combination
with Droplet Digital PCR (MSRE ddPCR) is a relatively newtechnique (Nell et al. 2020, Wang et al. 2021, van Zogchel et al.
2021, van de Leemkolk et al. 2022). Advantages for this method
include negligible DNA degradation and a minimum amount of
required starting material. These parameters make this method
well suited for applications that require liquid biopsy samples.
Additionally, because the DNA is preserved, other genetic
alterations such as copy number variants and single nucleotide
variants can be performed on the same sample.

Ch1+Ch2+:420, Ch1+Ch2–:3851, Ch1–Ch2+:2306, Ch1–Ch2–:340439
12,000Channel 1 amplitude

10,000

8,000

6,000

4,000

2,000

0
0

1,000

2,000

Methylation-independent primer forward		
Methylation-independent primer reverse		
Methylated probe			
Unmethylated probe			

3,000
4,000
Channel 2 amplitude

5,000

6,000

7,000

AGTtAGAGAGTGAtATGGACAACACCCTCTACTCTCTFAM-TTGGACGtATGCGTAGG
HEX-TTGGAtGtATGtGTAGGGAG

Fig. 9. Two-dimensional plot showing the duplex reaction for rat SNRPN
promotor from a mixture of methylated and unmethylated control samples.
A, methylation-specific primers (target: chr1:111592360-111593159 [reverse
complement]); B, methylation-independent primers (target rn7| chr1:111123593111123731). Chr, chromosome.

© 2023 Bio-Rad Laboratories, Inc.

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DNA Methylation Detection Using Droplet Digital PCR

References
Cho NY et al. (2020). Blood-based detection of colorectal cancer using cancerspecific DNA methylation markers. Diagnostics (Basel) 11, 51.
Hagelgans A et al. (2017). Identification of CpG sites of SERPINA5 promoter with
opposite methylation patterns in benign and malignant prostate cells. Anticancer Res
37, 6,609–6,618.
Jensen SØ et al. (2019). Novel DNA methylation biomarkers show high sensitivity
and specificity for blood-based detection of colorectal cancer—a clinical biomarker
discovery and validation study. Clin Epigenetics 11, 158.
Larsen LK et al. (2018). Noninvasive detection of high grade prostate cancer by DNA
methylation analysis of urine cells captured by microfiltration. J Urol 200, 749–757.
Lehmann-Werman R et al. (2018). Monitoring liver damage using hepatocyte-specificmethylation markers in cell-free circulating DNA. JCI Insight 3, e120687.
Nell RJ et al. (2020). Quantification of DNA methylation independent of sodium
bisulfite conversion using methylation-sensitive restriction enzymes and digital PCR.
Human Mutat 41, 2,205–2,216.
van de Leemkolk FEM et al. (2022). Quantification of unmethylated insulin DNA using
methylation sensitive restriction enzyme digital polymerase chain reaction. Transpl
Int 35, 10167.
Van Wesenbeeck L et al. (2018). Droplet digital PCR is an accurate method to assessmethylation status on FFPE samples. Epigenetics 13, 207–213.
van Zogchel LMJ et al. (2021). Novel circulating hypermethylated RASSF1A ddPCR
for liquid biopsies in patients with pediatric solid tumors. JCO Precis Oncol 5,
PO.21.00130.
Wang D et al. (2021). Development of a liquid biopsy based purely quantitative digital
droplet PCR assay for detection of MLH1 promoter methylation in colorectal cancer
patients. BMC Cancer 21, 797.

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