ANALYTICAL
BIOCHEMISTRY
Analytical Biochemistry 326 (2004) 278–280
Notes & Tips
Bisulfite conversion of genomic DNA for methylation
analysis: protocol simplification with higher recovery applicable
to limited samples and increased throughput
Victoria L. Boyd* and Gerald Zon
Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404, USA
Received 23 September 2003
Methylation of DNA as 5-methylcytosine (5mC)1 at
CpG dinucleotides in promoter regions of genomic
DNA (gDNA) is associated with silencing of tumor
suppressor genes and considered an important epige-
netic hallmark of cancer [1]. Various techniques for the
analysis of 5mC in gDNA have been reported [2–5], and
of these the most widely employed use the sodium bi-
sulfite method [3–7] for selective conversion of cytosine
(C) to uracil (U) without significant transformation of
5mC to thymine (T). Detection of U vs 5mC in the re-
sultant DNA sample can then be achieved by differential
hybridization, sequencing, or other techniques [2–6].
Notwithstanding the proven utility of this bisulfite
conversion method, it is commonly regarded both te-
dious and problematic to troubleshoot because of the
number of processing steps required and the relatively
complex chemistry issues involved. Moreover, a large
percentage of the gDNA sample is reported [6] to be
degraded during bisulfite treatment due to oxidative
damage and depurination under the required acidic
conditions and prolonged heating. While most protocols
typically recommend that 1 lg or more of gDNA be
used for the bisulfite conversion step, the amount of
recovered sample oftentimes allows only a few assays to
be performed even with amplification by PCR. To
overcome these and other limitations, we have exten-
sively investigated various aspects of this bisulfite
method. The results described below indicate that sam-
ple recovery can be significantly improved over previous
*
Corresponding author. Fax: +1-650-572-2743.
E-mail address: boydvn@appliedbiosystems.com (V.L. Boyd).
1
Abbreviations used: 5mC, 5-methylcytosine; gDNA, genomic
DNA; C, cytosine; U, uracil; T, thymine; (TE), Tris–EDTA; SAP,
shrimp alkaline phosphatase; exo, exonuclease 1; MSP, methylation-
specific PCR.
0003-2697/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.ab.2003.11.020
www.elsevier.com/locate/yabio
methods. The following bisulfite protocol featuring a
novel DNA isolation method provides a simplified,
high-recovery procedure that lends itself to increased
throughput and can be applied to 3000-fold less
sample (300 pg) than that which is typically recom-
mended. These improvements should be especially use-
ful for the growing number of clinical applications [8] of
methylated DNA analysis that could involve large
numbers and/or limited amounts of specimens.
gDNA from immortalized cells was purchased from
the Coriell cell repository (http://locus.umdnj.edu), and
fully methylated control DNA was purchased from
Serologicals (Norcross, GA). Three hundred nanograms
of purified gDNA or control DNA was bisulfite treated
as previously described [9] except for the following
modifications. The reaction was scaled to a final volume
of 200 lL, heated at 50 °C for 15 h in a microfuge tube,
without a layer of mineral oil, using a thermal cycler,
and then purified as follows.
(1) Dilute the bisulfite reaction with water to a total vol-
ume of 350–450 lL.
(2) Transfer this solution to an assembled Microcon 100
device (Millipore, Billerica, MA).
(3) Centrifuge at 2800 rpm (Eppendorf 5414) for 8–
10 min at 500g maximum, according to the manufac-
turer.
(4) Discard filtrate, add 350 lL of water to the upper
chamber, and centrifuge 6–8 min at 2800 rpm.
(5) Repeat step 4.
(6) Discard filtrate, add 350 lL of 0.1 M NaOH to the
upper chamber, and centrifuge at 2800 rpm for 6 min
(in situ desulfonation).
(7) Discard filtrate, add 350 lL of water to the upper
chamber, and centrifuge at 2800 rpm for 6-8 min,
leaving a small amount of water in the upper cham-
ber or keeping it damp.
 Notes & Tips / Analytical Biochemistry 326 (2004) 278–280
(8) Elute the sample by adding 50 lL of 1x Tris–EDTA
(TE) buffer; use the pipet for mixing and let stand in
column for 5 min.
(9) Invert the device and collect TE solution of the bi-
sulfite-converted DNA in a clean, labeled Eppendorf
tube.
The above procedure was initially developed using
bisulfite reagents freshly prepared from stock chemicals,
as is usually recommended in the literature [5,6,9] to
ensure effective conversion and high recovery. Signifi-
cantly, we found equivalent efficiency of the bisulfite
conversion (vide infra) using a much more convenient,
commercially available kit (Zymo Research, Orange,
CA). However, there are caveats: (1) no more than
300 ng of gDNA could be successfully used with the
manufacturerÕs recommended final reaction volume of
150 lL and (2) the Zymo resin purification cartridge
supplied with the reagent kit could not be successfully
used in place of the Microcon 100 cartridge in the
presently reported protocol.
For PCR, 0.5 lL (3 ng assuming 100% yield) of the
bisulfite-converted DNA was used in a 20-lL reaction
prepared with 2x AmpliTaq Gold PCR Master Mix
(Applied Biosystems, Foster City, CA) containing
250 nM each of the forward and reverse primers which
were selected (see [10] and http://itsa.ucsf.edu/~urolab/
methprimer) such that they flanked the CpG region of
interest. Thermal cycling conditions were 5 min at 95 °C,
40 cycles of 95 °C/30 s, 60 °C/45 s, 72 °C/1 min; stored at
4 °C. By use of a fluorescently (ABI FAM dye)-labeled
primer, PCR amplicons were directly analyzed on an
ABI PRISM 310 Genetic Analyzer using POP-4 poly-
mer and run module GS POP4 (1 mL) A.
Methylation-specific PCR (MSP) was also successful
using 3 ng of the bisulfite-converted DNA with the
PCR conditions described above and with only 30
cycles of thermal cycling. Eight published MSP primer
sets, with one FAM-labeled primer, were tested. All
gave PCR amplicons of the correct sizes, as expected
for the fully methylated and unmethylated samples. A
signal strength of about 500 FU was obtained when a
1/10 dilution of the PCR was added into formamide
and analyzed on an ABI PRISM 310 Genetic
Analyzer.
Prior to sequencing the PCR amplicons, the residual
primers and dNTPs were removed by treatment of a 4-
lL aliquot of the above PCR mixture with an equal
volume mixture containing 2 units each of shrimp al-
kaline phosphatase (SAP) and exonuclease 1 (exo) (USB
Corp., Cleveland, OH). The reaction was incubated at
37 °C for 1 h, and then heat-denatured at 75 °C for
15 min. A 4-lL aliquot of the exo/SAP reaction was
added to a solution containing 1–4 lL of BigDye Ter-
minator v1.1 cycle sequencing reaction mix (Applied
Biosystems), 2 lL of BigDye Terminator v1.1 5x
sequencing buffer (Applied Biosystems), 2 lL of the
279
reverse PCR primer (5 lM), and enough water for a final
volume of 20 lL. Thermal cycling conditions were 95 °C/
1 min, 30 cycles of 96 °C/10 s, 52 °C/10 s, 60 °C/4 min;
stored at 4 °C. The purified (Performa 96-well plate;
Edge Biosystems, Gaithersburg, MD) sequencing
products were analyzed using an ABI PRISM 3700
DNA Analyzer or Applied Biosystems 3730 DNA
Analyzer with KB basecaller.
The generally accepted [5] multistep mechanism for
bisulfite conversion of C to U involves (1) reversible
addition of the sulfite moiety to the 6-position of C (pH
5), (2) irreversible hydrolysis (deamination) to form a
sulfonated derivative of U, and (3) elimination of bi-
sulfite from the sulfonated U derivative under basic
conditions. A single treatment of a bisulfite-treated
sample in the Microcon 100 cartridge with 0.1 M NaOH
led to high recovery of DNA, presumably due to in situ
desulfonation leading to easier extraction into the TE
elution buffer. Analysis of serial dilutions of a gDNA
sample demonstrated that, using the presently reported
protocol, as little as 300 pg could be successfully bi-
sulfite-converted and isolated in a final volume of 10 lL
to provide as many as 10 30-pg (assuming 100% yield)
PCR assays each representing 20 copies of gDNA.
Compared to 1 lg of gDNA typically recommended,
our ability to use only 300 pg of gDNA represents an
3000-fold improvement in the amount of starting
sample that can be processed to afford enough converted
DNA for a reasonably large number of subsequent as-
says (10 in this example).
To validate the presently reported protocol, we ana-
lyzed by the above methods (data not presented) a
number of previously reported [11] tumor suppressor
gene targets that show correlations between methylation
status and gene expression (or cancer). As expected, C
was present in CpGs only in the fully methylated control
DNA whereas gDNA from immortalized cells obtained
from Coriell was unmethylated in most regions but
methylated at CpGs in some of the gene promoter re-
gions. Robustness of the protocol was indicated by our
ability to successfully sequence all 25 gene targets
without any failed reactions or requiring repetition. In
conclusion, it is also worthwhile to note that several
independent laboratories that routinely use previously
published bisulfite-conversion procedures, or variants
thereof, have evaluated the presently reported protocol
and each found that it was readily implemented and
offered advantages.
Acknowledgments
We thank Professor Jack Richards of the California
Institute of Technology for his continued interest in this
work and Ms.Cheryl Heiner and Ms. Julie Zhao for
technical advice with regard to DNA sequencing.
280 Notes & Tips / Analytical Biochemistry 326 (2004) 278–280
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