Bisulfite Conversion Kit(version A1)

Catalog No. 55016

Active Motif North America
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Copyright 2013 Active Motif, Inc.

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Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of any
kind, either explicit or implied. Information may be changed or updated in this manual at any time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation is
proprietary information and protected by the copyright laws of the United States and international treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2013 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respective
companies.
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TABLE OF CONTENTS

Page

Overview  . . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Flow Chart of Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Introduction  . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Traditional Methods to Study DNA Methylation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
References . . . . .  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .5
Kit Performance and Benefits  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Kit Components and Storage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Protocols
Buffer Preparation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Bisulfite Conversion Kit Protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section A: Proteinase K Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section B: Conversion Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Section C: On-Column Desulfonation and DNA Purification . . . . . . . . . . . . . . . . . . . . . . . . 12
Optional Downstream Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section D: Quantification of Bisulfite Converted DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section E: Conversion-Specific PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Section F: Agarose Gel Analysis of PCR Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  14
Section G: DNA Clean Up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Section H: DNA Cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  16
Section I: Analyzing the Sequencing Results  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Appendix
Section J. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  18
Section K. Related Products  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  19
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

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Overview
For the investigation of DNA methylation patterns, bisulfite conversion and subsequent DNA
sequencing is the gold standard because it provides detailed information on the methylation
pattern of individual DNA molecules at single-base-pair resolution. Bisulfite conversion is basedon the deamination of cytosine residues into uracils in the presence of sodium bisulfite. Since
methylated cytosine residues (both 5-mC and 5-hmC) are not converted under these conditions,the original methylation state of the DNA can be determined. Converted DNA can be analyzed
by PCR amplification followed by DNA sequencing (gene-specific or genome-wide), array-based
methods, or other downstream applications.
Active Motif’s Bisulfite Conversion Kit simplifies analysis of DNA methylation by providing optimized conversion reagents, an easy-to-use protocol and a positive control conversion-specific PCRprimer pair that is specific for bisulfite-converted human or mouse DNA. Because the primer pair
produces a PCR product only if conversion has occurred, you can confirm the procedure worked
before starting sequencing or other analysis methods.
In the Bisulfite Conversion Kit method, DNA of interest is rapidly heat denatured in a thermocycler
in the presence of the bisulfite conversion reagent and a DNA denaturant. The conversion reaction
can be performed is as little as 1.5 hours. After DNA conversion, the sample is added to the included DNA purification columns, and a simple, on-column desulfonation is performed. Ready-touse converted DNA is then eluted from the columns. For your convenience, the included positive
control conversion-specific PCR primer pair can be used to assess the success of the bisulfite
conversion before DNA sequencing because the included primers only anneal to converted humanor mouse DNA.

product

formatcatalog no.

Bisulfite Conversion Kit

50 rxns

55016

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1Flow Chart of Process
Sulfonation

Cytosine

Deamination

Cytosine-SO3–

Desulfonation

Uracil-SO3–

UracilSchematic of the bisulfite conversion reaction of unmethylated cytosine to uracil.
The first step in the bisulfite conversion reaction is sulfonation, where sodium bisulfite is added to the double bond ofcytosine for form a cytosine-bisulfite derivative. This reaction is reversible, with the forward reaction being favored by hightemperature and low pH. The second step is an irreversible hydrolytic deamination of the cytosine-bisulfite derivative that
results in a uracil-bisulfite derivative. This reaction is also favored by low pH. The final step involves desulfonation of theuracil-bisulfite to uracil under high pH conditions. Only unmethylated cytosines are susceptible to the bisulfite reaction.
Methylated (5-mC and 5-hmC) cytosines do not undergo conversion.

Genomic
DNA

Proteinase K
treatment (30 min)DNA conversion
in thermocycler (1.5 h)

Add DNA
Binding Buffer

Load Spin Column

Elute

Wash

Add Desulfonation Buffer

Wash

Bisulfite
converted
DNA

Flow chart of the Bisulfite Conversion Kit.
In the Bisulfite Conversion Kit, genomic DNA of interest is proteinase K treated, then rapidly heat denatured in a thermocycler in the presence of the bisulfite conversion reagent and DNA denaturant. Following a 1.5 hour DNA conversion reaction,
the sample is added to the included DNA purification columns, and a simple on-column desulfonation is performed. DNA
is then eluted from the column and is ready for use in PCR.

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Introduction
DNA methylation is an essential epigenetic modification that plays a key role in transcriptional
regulation and assures the proper regulation of gene expression and stable gene silencing innormal cells. DNA methylation is the covalent addition of a methyl group to the fifth positionof the cytosine pyrimidine ring in DNA and usually occurs within CpG dinucleotides, although insome cases the DNA methylation can also be found in a non-CpG context1, 2. CpG dinucleotides
are concentrated in large clusters called CpG islands, which are often positioned in the promoter
regions of many genes and are usually unmethylated3. It is estimated that the human genome
contains around 29,000 CpG islands and that almost 60% of all human genes are associated with
CpG islands4. Aberrant cytosine methylation is associated with silencing of tumor suppressor
genes5 and plays a decisive role in the development of many cancers6-14. Alterations in the methylation status of DNA are promising candidates for highly specific and sensitive indicators of cancer
diagnosis and prognosis15-17. Apart from carcinogenesis, DNA methylation is crucial for a variety ofprocesses, such as genomic imprinting, X-chromosome inactivation, and suppression of repetitiveelements4, 18. Thus profiling DNA methylation across the genome is vital to understanding the influence of epigenetics.
The bisulfite reaction was first described in the early 1970s19 and was used by Frommer et al.20,
and Clark et al.21 to distinguish between cytosine and 5-methylcytosine (5-mC) in DNA. It is now
known that both 5-methylcytosine and 5-hydroxymethylcytosine (5-hmC) remain unchanged
during conversion and therefore this method cannot be used to distinguish between the different
methyl modifications22-24. In the bisulfite conversion reaction, DNA is first treated with sodium
bisulfite to convert cytosine residues into uracil in denatured (single-stranded) DNA, underconditions whereby 5-mC and 5-hmC remain essentially non-reactive. The DNA is then amplified
by PCR where the uracils are converted to thymines. Bisulfite converted DNA can be analyzed
for gene- or allele-specific methylation patterns, or adapted for genome-wide DNA methylation
analysis. A methylation profile of the sample can then be created by comparing the sequence ofthe converted DNA to untreated DNA.
Active Motif’s Bisulfite Conversion Kit simplifies analysis of DNA methylation by providing
optimized conversion reagents and an easy-to-use protocol. The conversion reaction can be
performed in as little as 1.5 hours with a 99% conversion efficiency of unmethylated cytosines touracils. The Bisulfite Conversion Kit includes a positive control conversion-specific PCR primer pairthat is specific for bisulfite-converted human and mouse DNA. Because the primer pair produces a
PCR product only if conversion has occurred, you can validate the success of the conversion reaction before spending extra time and money on sequencing.

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Traditional Methods to Study DNA Methylation
To date, there are several methods used for methylation analysis:
1. Methylation-sensitive restriction enzyme analysis: Isoschizomers of bacterial restriction
endonucleases with different sensitivities for methylated DNA can be used to determine
the methylation status of specific CpG-dinucleotides25. Pairs of isoschizomers are used thatrecognize the same restriction site, but one enzyme is unable to cleave methylated DNA.
Methylation-sensitive restriction enzymes have several limitations including that methylation-sensitive restriction merely informs on the methylation status of the cytosine residues
which are recognized by the restriction enzymes used.
2. Bisulfite conversion: Bisulfite conversion19-21 consists of the treatment of genomic DNA
with sodium bisulfite, leading to deamination of unmethylated cytosines into uracil. PCR
is then performed with primers that differentiate between methylated and unmethylatedsequences. Bisulfite converted DNA can be sequenced in order to quantify the level of DNA
methylation in the sample. However, bisulfite-based techniques do involve chemical treatments that can damage DNA and limit throughput. Additionally, PCR primer design becomesdifficult due to reduction in genome complexity after bisulfite treatment.
3. Methylated DNA Immunoprecipitation (MeDIP): In this assay, an antibody specific formethylated cytosines (anti-5-methylcytosine antibody or anti-5-hydroxymethylcytosine antibody) is used to immunoprecipitate methylated DNA from genomic DNA fragmented by enzymatic digestion or sonication26. The resulting enrichment is usually analyzed by PCR based
methods; thus MeDIP can be combined with DNA microarrays or adapted with sequencing
primers for genome-wide analysis of CpG methylation. However, this technique is relatively
time-consuming and requires a large amount of fragmented DNA starting material and does
not give single-base-pair resolution.
4. Methyl-CpG Binding proteins: This family of proteins takes its definition from themethyl-CpG binding domain (MBD), the minimum portion with specific affinity for a single,
symmetrically methylated CpG pair. The MBD2b protein has been found to possess one of
the highest affinities for methylated DNA among MBD proteins and has the greatest capacity
to differentiate between methylated and unmethylated DNA27. The Methylated CpG Island
Recovery Assay (MIRA) utilizes the combination of MBD2b with its binding partner MBD3L1,
methyl-CpG-binding protein 3-like-1, to generate a higher affinity for methylated DNA than
MBD2b protein alone28,29. Methyl-CpG binding proteins are limited to the evaluation of
methylated DNA in a CpG context as the proteins do not recognize methylated cytosinesthat exist outside of a CpG dinucleotide. The MBD2 proteins are only capable of binding to
5-mC methylation; they cannot be used to enrich for 5-hmC methylation24.

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References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.

Ramsahoye, B.H. et al. (2000) Proc. Natl Acad. Sci. USA, 97: 5237-5242.
Lister, R. et al. (2009) Nature, 462: 315-322.
Antequera, F. and Bird, A. (1993) Proc. Natl Acad. Sci. USA, 90: 11995-11999.
Bird, A. (2002) Genes Dev, 16: 6-21.
Issa, J.P. (2004) Nat Rev Cancer, 4: 988-993.
Toyota M., et al. (1999) Proc. Natl Acad. Sci. USA, 96: 8681-8686.
Hughes, L.A. et al. (2012) Biochim Biophys Acta., 1825: 77-85.
Samowitz, W.S. (2007) Mol Diagn., 9: 281-283.
Marsit, C.J. et al. (2006) Cancer Res., 66: 10621-10629.
Suzuki, M. et al. (2006) Cancer, 106: 2200-2207.
Shen, L. et al. (2002) J Natl Cancer Inst., 94: 755-761.
Fang, F. et al. (2011) Sci Transl Med., 3:75ra25.
Jones, P.A. and Baylin, S.B. (2002) Nature Rev. Genet., 3: 415-428.
Esteller, M. et al. (2001) Cancer Res., 61: 3225-3229.
Laird, P.W. (2003) Nat Rev Cancer, 3: 253-266.
Van Engeland, M. et al. (2011) J Clin Oncol., 29: 1382-1391.
Esteller, M. (2008) N Engl J Med., 358: 1148-1159.
Reik, W. et al. (2001) Science, 293: 1089-1093.
Shapiro, R. et al. (1974) J Am Chem Soc, 96(3): 206-212.
Frommer, M. et al. (1992) Proc. Natl Acad. Sci. USA, 89: 1827-1831.
Clark, S.J. et al. (1994) Nuc. Acids Res., 22: 2990-2997.
Huang, Y. et al. (2010) PLoS One, 26: e8888.
Jun, S. et al. (2010) Nucl. Acids Res., 38: e125.
Nestor, C. et al. (2010) BioTechniques, 48: 317-319.
Dalh, C. and Guldberg, P. (2003) Biogerontology, 4: 233-250.
Weber, M. et al. (2005) Nat. Genet., 37: 853-832.
Fraga, M.F. et al. (2003) Nucleic Acids Res., 31: 1765-1774.
Rauch, T. and Pfeifer, G. (2005) Lab. Investigation, 85: 1172-1180.
Jiang, C.L. et al. (2004) J. Bio. Chem., 279: 52456-52464.

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5Kit Performance and Benefits
The Bisulfite Conversion Kit is for research use only. Not for use in diagnostic procedures.
Sensitivity: Bisulfite Conversion can be performed on 200 pg - 2 µg of DNA, with 500 ng to 2 µg
optimal.
Conversion efficiency: >99%
DNA Recovery: >80%

Me

Me

Me

GATCGACGATCGGAGCGTAGGTACGACGTT
Bisulfite Conversion

GATCGAUGATCGGAGUGTAGGTACGAUGTTPCR Amplification

GATCGATGATCGGAGTGTAGGTACGATGTT
Figure 1: DNA sequence following bisulfite conversion and PCR amplification.
This figure shows an example of the effect of bisulfite treatment on a DNA sequence. The original DNA sequence contains
6 cytosine residues, 3 methylated (Me) and 3 unmethylated. Following bisulfite conversion the unmethylated cytosines areconverted into uracil while the methylated cytosines remain as cytosine. The methylation profile of the DNA can then be
determined by PCR amplification followed by DNA sequencing, where the uracils will be detected as thymine. This is why it
is often stated that bisulfite treatment converts C to T.

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Kit Components and Storage
Bisulfite Conversion Kit components arrive at room temperature and can be stored at 4ºC prior to
first use. Then, we recommend storing each component at the temperatures listed in the table below. Components are guaranteed stable for 6 months from date of receipt when stored properly.
Reagents

Quantity

Storage

Conversion Reagent

5 tubesRT

Denaturation Reagent

875 µlRT

Hydroquinone

5 x 100 µl

RTBuffer A

1.75 ml

RT

Buffer B

1.1 mlRT

DNA Binding Buffer

2 x 15 ml

RT

DNA Wash Buffer

25 ml

RT

DNA Elution Buffer

2.5 mlRT

DNA purification columns

50 ea

RT

DNA column collection tubes

50 ea

RT

Proteinase K

2 mg

-20°C

Conversion-specific PCR primer pair (4 µM)

200 µl-20°C

Additional Materials Required
Bisulfite Conversion and PCR Analysis• Purified sample DNA• 0.2 ml PCR tubes and caps
• 100% isopropanol• Microcentrifuge tubes and microcentrifuge• dNTP mixture (10 mM each)• Thermocycler• Hot Start Taq polymerase (5 U/µl) and PCR reaction buffer recommended for use withbisulfite converted DNA (e.g. Qiagen HotStarTaq DNA polymerase, Catalog No. 203203)• Methylation-specific PCR primers for the gene of interest• 2% agarose TAE gel• Gel apparatus and power supply• 6x Loading dye• Molecular weight marker
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DNA Clean Up• (Optional) DNA purification kit (e.g. QIAquick PCR purification kit, Qiagen Cat. No. 28104)• Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v/v)• 3 M Sodium acetate, pH 5.5• Glycogen, molecular biology grade (20 ng/µl) or tRNA (10 µg/µl)• 100% ethanol
DNA Cloning and Sequencing• Ligation vector (e.g. Topo TA Cloning Kit for sequencing, Life Technologies Cat. No. 450030)• Competent E.coli cells (e.g. RapidTrans™ TAM 1, Active Motif Cat. No. 11096)• Bacterial growth media and plates with appropriate selection• DNA miniprep materials• DNA sequencing reagents

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8

Protocols
Buffer Preparation
Input DNA
Input DNA should be in the range of 200 pg to 2 µg, with the optimal range being 500 ng to2 µg. Please see the Appendix A: Troubleshooting section for suggestions on adding a carrier
when using less than 100 ng of DNA.
Preparation of Proteinase K working solution
To improve the efficiency of the bisulfite conversion reaction the Input DNA is treated with Proteinase K prior to performing the conversion reaction. Proteinase K is supplied lyophilized and canbe stored at 4ºC prior to first use. Resuspend 2 mg of Proteinase K in 100 µl sterile water. Vortex
to fully resuspend. The final concentration of the Proteinase K working solution is 20 mg/ml andshould be stored at -20°C.
Preparation of Conversion Buffer
Five tubes of Conversion Reagent are provided in the Bisulfite Conversion Kit. Each vial is sufficientfor performing 10 bisulfite conversions. Prepare the Conversion Buffer by resuspending one of theConversion Reagent tubes with 700 µl dH2O, 350 µl Buffer A and 175 µl Denaturation Reagent. Mix
at room temperature for 10 minutes with intermittent vortexing.
NOTE:

This is a saturated solution and it is normal for the Conversion Reagent to notdissolve completely. The Conversion Buffer is stable for 1 week at -20°C.
Conversion Reagent is sensitive to air and moisture. Keep cap on tight and re-cap
immediately after use.

Preparation of Hydroquinone
Hydroquinone is an alkalizing agent and prevents DNA from strand breakage because of
depurination. Each tube provided can be used for 10 conversion reactions. Prepare a working stockof Hydroquinone by resuspending one tube with 100 µl dH2O. Keep mixture in dark and discard
after use.
Preparation of Desulfonation Buffer
For each reaction, combine 22 µl Buffer B with 88 µl dH2O and 110 µl 100% isopropanol. If thisreagent will not be used immediately, it can be stored for up to one week at -20°C.
DNA Binding Buffer
Is supplied ready to use.
DNA Wash Buffer
Is supplied ready to use.
DNA Elution Buffer
Is supplied ready to use.
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Positive control Conversion-specific PCR primer pair
The Bisulfite Conversion Kit’s positive control Conversion-specific PCR primer pair (4 µM) is specific for amplification of bisulfite converted human or mouse DNA. This primer pair should amplify
DNA that has been successfully bisulfite converted to generate a 220 bp PCR product. It should
not generate a PCR product with untreated human or mouse DNA. If using a different species ofDNA besides human or mouse, the included positive control Conversion-specific PCR primer pair
will not work and conversion-specific primers will need to be designed to your species of interest.
Design of PCR primers
PCR primer design is critical for successful analysis of converted DNA. There are several sitesthat offer information on designing methylation-specific PCR primers. The MethPrimer website:
http:// www.urogene.org/methprimer/ is a free site that contains several useful tools for thedesign of bisulfite-conversion-based Methylation PCR Primers. Additional programs include,
MethylViewer http://dna.leeds.ac.uk/methylviewer/ and PREMIER Biosoft International offers the
Beacon Designer program http://www.premierbiosoft.com/crm/jsp/com/pbi/crm/ clientside/
ProductList.jsp. We suggest designing both bisulfite conversion specific and untreated PCR primer
pairs for each gene of interest to validate the success of the conversion reaction prior to downstream analysis. For design of primers to be used in the same PCR reaction as the positive control
Conversion-Specific PCR primer pair we recommend an annealing temperature between 56-60°C.

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Bisulfite Conversion Protocol
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
The Bisulfite Conversion Kit can efficiently convert DNA samples containing between 200 pg to2 µg of DNA, with the optimal input range of 500 ng to 2 µg. The positive control Conversionspecific PCR primer pair is designed to anneal to bisulfite converted human and mouse DNA.
If you plan to perform the positive control reactions, it is recommended that you perform theconversion reaction using 500 ng of human or mouse DNA.

Step A: Proteinase K Treatment1.

Add sample DNA to a microcentrifuge tube for proteinase K treatment. Treatment withproteinase K can be performed in large scale using the entire mass of DNA recovered fromsample extraction. Alternatively, for smaller scale treatment, add the appropriate amount of
DNA that will be used for bisulfite conversion to the microcentrifuge tube.

2.

Add Proteinase K working solution (20 mg/ml) to the DNA to achieve a final concentrationof 50 µg/ml. (For example if using 1 ml DNA, add 2.5 µl Proteinase K working solution.) If thevolumes needed are too small to accurately pipet, prepare an additional 1:10 dilution of theProteinase K working solution in sterile water just before use to make a 2 mg/ml stock.

3.

Incubate the DNA with Proteinase K at 50°C for a minimum of 30 minutes. Longer incubation
times, including overnight, may also be performed at 50°C.

Step B: Conversion Reaction
1.

Prepare Conversion Buffer and Hydroquinone as described in the Buffer Preparation Section
on page 9.

2.

Set up a conversion reaction for each sample by adding the reagents in the order listed
below into PCR tubes. Mix well by pipetting. The final volume should be 140 µl. If using lessthan 13 µl of DNA, adjust the volume with water to a final volume of 140 µl. Keep hydroquinone away from light.
Reagent

Quantity

DNA

13 µl

dH2O

X µl

Hydroquinone

7 µl

Conversion Buffer

120 µlTotal Volume

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3.

Place tubes in the thermocycler and set the following program. To prevent evaporation ofsamples, use the heated lid or overlay reactions with mineral oil.

		

95°C for 30 seconds
58°C for 20 minutes(95°C for 10 seconds, 58°C for 20 minutes) for 3 cycles
Hold at 4°C

Note:

4.

The suggested 1.5 hour conversion PCR conditions are ideal for undigested genomic DNA. The number of cycles can be increased if incomplete conversion is aconcern, however, incubation periods longer than 3 hours are not recommended.
If using fragmented DNA, or large quantities of genomic DNA, please refer toAppendix Section A: Troubleshooting Guide on page 18 for alternative bisulfite
conversion conditions.

Remove tubes and continue with Step C below. The protocol can also be stopped here andDNA samples can be kept at 4°C in the dark for up to 5 days.

Step C: On-column Desulfonation and DNA Purification1.

Prepare Desulfonation Buffer as described in the Buffer Preparation section on page 9.

2.

For each conversion reaction, aliquot 500 µl DNA Binding Buffer into a 1.5 ml microcentrifuge
tube. Add the entire conversion reaction mixture to the DNA Binding Buffer in the tube andvortex.

3.

Remove the desired number of DNA purification columns and place each one in a collection
tube.

4.

Pipet each DNA Binding Buffer/conversion reaction mixture into one of the DNA Purification
columns and spin at 10,000 rpm for 30 seconds in a microcentrifuge. Empty the collection
tube and place the column back on the collection tube.

5.

Add 200 µl DNA Wash Buffer to each column and spin at 10,000 rpm for 30 seconds in a
microcentrifuge.

6.

Add 200 µl Desulfonation Buffer to each column and incubate 20 minutes at room temperature. Spin at 10,000 rpm for 30 seconds in a microcentrifuge. Empty the collection tube andplace the column back on the collection tube.

7.

Add 200 µl DNA Wash Buffer to each column and spin at 10,000 rpm for 30 seconds in a microcentrifuge. Empty the collection tube and place the column back on the collection tube.

8.

To remove any residual wash buffer, spin at 10,000 rpm for 30 seconds in a microcentrifuge.
Remove the column and place in a new 1.5 ml microcentrifuge tube.

9.

Add 30 µl DNA Elution Buffer directly to the filter of the column.

10. Incubate 3 minutes at room temperature. Spin at 10,000 rpm for 30 seconds in a microcentrifuge. The eluate will contain the converted DNA.
11.

The eluted DNA is now ready for downstream analysis.
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Optional Downstream Analysis
Step D: Quantification of Bisulfite Converted DNA
Following bisulfite treatment of DNA, the original base-pairing of DNA no longer exists since
the unmethylated cytosine residues are converted into uracil. If using a spectrophotometer, set
the unit to measure RNA absorption (using a conversion of 40 µg/ml for A260 nm = 1.0) because
bisulfite converted DNA resembles RNA since it contains uracil and is largely single stranded.
Alternatively, use a Qubit ssDNA Assay or check the quality of the bisulfite converted DNA using
Agilent Bioanalyzer with RNA 6000 Pico Kit or similar detection methods. However, if the bisulfite
DNA is PCR amplified prior to quantification, traditional methods to quantify DNA can be used.

Step E: Conversion-Specific PCR
Determine the number of conversion-specific PCR reactions to perform. The included positive
control Conversion-specific PCR primer pair can be used to quickly assess the success of thebisulfite conversion reaction using human or mouse DNA samples. For conversion-specific gene
analysis, use a conversion-specific PCR primer pair and an untreated PCR primer pair (if desired)for each gene locus of interest. Follow the recommendation listed on page 10 to design bisulfite
conversion-specific primers for your gene of interest. We recommend an annealing temperature
between 56-60°C if the primers are to be used in the same PCR reaction as the positive control
Conversion-Specific PCR primer pair.
We also recommend performing a Hot Start PCR reaction when working with bisulfite converted
DNA to ensure reliable and consistent amplification of the DNA template.

Positive Control
Conversion-specificPrimer Pair

Gene-specificConverted
Primer Pair

Gene-specific
Untreated
Primer Pair

Bisulfite Converted DNA

Rxn 1

Rxn 4

Rxn 7

Untreated DNA

Rxn 2

Rxn 5

Rxn 8

H2O control

Rxn 3

Rxn 6

Rxn 9

DNA Template1.

Label the desired number of PCR tubes and place in a PCR tube rack on ice.

2.

Make a 20 ng/µl dilution of unconverted genomic DNA (the same Input DNA that was used
for the conversion reaction).

3.

Prepare a PCR master mix for each of the primer sets to be tested in 1.5 ml microcentrifuge
tubes as described below. DNA templates will be added later directly to the PCR tubes.
Combine components on ice and mix by vortexing. Four reactions are prepared to accountfor any loss during pipetting. The table below is provided as an example, please adjust as
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needed to follow the recommendation of the Hot Start PCR polymerase to be used.
Reagent

One Rxn

Master Mix

dH2O

13.55 µl54.2 µl

PCR primer pair (4 µM)

2 µl

8 µl

dNTP mixture (10 mM each dNTP)

0.25 µl

1 µl

10X PCR Buffer

2 µl

8 µl

Hot Start Taq (5 U/µl)

0.2 µl

0.8 µlTotal Volume (Not including DNA template)

18 µl

72 µl

4.

Aliquot 18 µl of this PCR cocktail into the appropriately labeled tubes on ice.

5.

Add template DNA or water to the PCR tubes as indicated below and mix. Ensure that theentire 20 µl PCR reaction is on the bottom of the tube. Centrifuge tubes if necessary.

6.

PCR Rxn #PCR Master Mix

DNA Template

dH2O

1, 4 & 7

18 µl

2 µl Bisulfite Converted DNA–

2, 5 & 8

18 µl

2 µl Untreated DNA (20 ng/µl)–

3, 6 & 9

18 µl

–

2 µl

Place tubes in PCR cycler and program the thermocycler as below. (To prevent evaporation ofsamples, use the heated lid or overlay reactions with mineral oil.)
94°C for 15 minutes(94°C for 30 seconds, 58°C for 30 seconds, 72°C for 1 minute) for 30–35 cycles
72°C for 10 minutes
Hold at 4°C

Step F: Agarose Gel Analysis of PCR Products
1.

Pour a 2% agarose gel in 1X TAE.

2.

Add 5 µl from each of the PCR reactions to 1 µl 6X DNA loading dye and load sample on a gel.
Use a 100 or 50 bp DNA ladder. Run the gel until the marker is near the bottom of the gel
and the markers are well separated. Only the PCR reactions containing bisulfite converted
DNA should show a PCR product when using conversion-specific primer sets. Untreated DNA
and the H2O control PCR reactions should not have any PCR amplification. For the positive
control Conversion-specific PCR primer pair, the bisulfite converted DNA should generate a
220 bp amplicon as shown in Figure 2 on the next page.

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Figure 2: Positive Control Conversion-specific PCR primer pair is specific for converted human or mouse DNA.
The figure above depicts the PCR results of human (A) and mouse (B) genomic DNA that was either bisulfite converted(Bis.) according to the assay protocol or untreated (Untr.) and amplified using the included positive control Conversionspecific PCR primer pair. The primer pair is specific towards bisulfite converted DNA and therefore a 220 bp PCR ampliconis present in the bisulfite treated DNA samples and not in the untreated DNA samples.

Step G: DNA Clean Up
Prior to cloning it is necessary to clean up the DNA. Use the following protocol to perform aphenol/chloroform extraction followed by ethanol precipitation. Alternatively, DNA can be purified using columns such as QIAquick PCR purification kit (Qiagen part no. 28104). Elute in 30-50 µl
volume. If more than one band is amplified by the PCR reaction in Step E, the DNA band of the
expected size should be gel-purified using a gel extraction kit such as QIAEX II gel extraction kit(Qiagen part no. 20021).
1.

To the remaining PCR reaction from Step E, add the necessary amount of sterile water tomake the total volume 100 µl.

2.

Add an equal volume of Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v/v) to the sample.

3.

Vortex the tube at maximum speed for 15 seconds.

4.

Centrifuge the tube for 5 minutes at 12,000 x g at room temperature.

5.

Carefully transfer the top aqueous phase to clean microcentrifuge tube without collecting
any of the lower organic phase or precipitate that may occur between the phases.

6.

To each sample add:
1 µl Glycogen (20 ng/µl)
1/10 sample volume of 3 M sodium acetate pH 5.5
2.5 sample volumes of 100% ethanol

7.

Mix well and incubate at -80°C for at least 2 hours.

8.

Centrifuge the tube for 20 minutes at 12,000 x g, 4°C.

9.

Carefully discard the supernatant without disturbing the pellet.

10. Add 500 µl of cold 70% ethanol.
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11.

Centrifuge the tube for 10 minutes at 12,000 x g, 4°C.

12.

Carefully discard the supernatant without disturbing the pellet.

13.

Air-dry the pellet for 5-10 minutes (do not completely dry the pellet).

14.

Resuspend the DNA pellet in 30 µl sterile DNase-free water.

15.

This eluted DNA can be used immediately in cloning or stored at -20°C. If the DNA is stored
at -20°C, it will need to be reheated at 37°C for 10 minutes prior to use to ensure DNA does
not stick to the walls of the tube.

Step H: DNA Cloning
1.

The cleaned PCR product can be cloned into a DNA vector of choice according to the
instructions of the manufacturer (e.g. Topo TA Cloning Kit for sequencing, Life Technologies
Cat. No. 450030). If possible, plasmids containing the LacZ gene for blue/white screening arerecommended. An example ligation reaction is shown below, but please follow the recommendations associated with the DNA vector of choice.
Reagent

Quantity

DNA template

4 µl

Ligation Buffer

1 µl

DNA vector

1 µl

Total Volume

6 µl

Incubate 5 minutes at room temperature.
2.

Transform competent bacterial cells, such as RapidTrans™ TAM 1 (Active Motif Cat. No.
11096), with 2 µl of the ligation reaction. Follow the appropriate protocol for the competentcells used. The RapidTrans™ TAM 1 chemically competent cell protocol is listed below as an
example.
a.

Remove required number of transformation reaction tubes from –80°C storage andplace on ice to thaw.

b.

Add 1-5 μl of plasmid DNA/ligation to thawed cells. Mix by tapping tubes gently andreplace on ice immediately. Do not mix by vortexing or pipetting. Do not add morethan 5 μl (10% of competent cell volume) to reaction.

c.

Incubate transformation reactions on ice for 30 minutes.

d.

Heat-shock the tubes by immersing in a 42°C water bath for exactly 30 seconds.

e.

Replace transformation reactions on ice for 2 minutes.

f.

Aseptically add 250 μl SOC medium to each reaction.

g.

Incubate tubes at 37°C for 1 hour with shaking at 225-250 rpm.

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h.

Using a sterile spreader, plate out 20-200 μl of each transformation on pre-warmed
LB agar plates. If using blue/white screening, ensure that the selective plate contains
X-Gal. For preparation of X-gal Stock solution and plates see note below.

Note:

Make a 40 mg/ml solution of X-gal in dimethylformamide (DMF). Use a glass or
polypropylene tube and wrap in aluminum foil to prevent damage by light. Store
at –20°C. Plate 40 μl of X-gal stock solution on top of the LB agar and let standfor approximately 1 hour. Alternatively, add the X-gal to the molten LB agar prior
to pouring plates.

i.

Allow plates to completely absorb any excess media.

j.

Incubate inverted plates overnight at 37°C.

3.

Select 10 colonies for screening. Select white colonies if using blue/white screening as theexpression of LacZ has been interrupted with the insertion of the ligation product.

4.

Isolate plasmid DNA of the 10 selected colonies using any commercially available kit or
published protocol.

5.

Analyze the isolated DNA using restriction enzyme analysis to confirm the presence of thecorrect insert, or by PCR using region-specific primers or primers corresponding to sequences
within the vector (e.g. M13 forward and reverse primers)

6.

Submit DNA from the positive clones for sequencing. Use forward and reverse primers basedon the DNA vector used for cloning. It is recommended to submit 10 colonies for sequencing to ensure representative results. It is also necessary to submit unconverted DNA for
sequencing comparison.

Step I: Analyzing the Sequencing Results
1.

If the CpG dinucleotide was methylated in the original DNA, the sequencing read will show
a CG at that location. If the CpG dinucleotide was unmethylated in the original DNA, thebisulfite conversion will have converted the cytosine to uracil and the sequencing read willshow a TG at that location.

2.

Compare the bisulfite converted sequence data against the unconverted DNA sequence.

3.

Quantify the number of methylation sites for the region of interest to determine the percentage of methylation.

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Appendix
Section J. Troubleshooting Guide
Problem/question

Recommendation

Using less than 100 ng of
DNA as starting material

When converting a small amount of DNA, add 1 µg Glycogen (20 ng/µl) or tRNA (10 µg/µl) as a carrier to the 500 µl DNA Binding Buffer for each reaction during the on-column
desulfonation and DNA purification step to minimize sample loss.

Starting DNA volume isgreater than 13 µl

DNA volumes up to 20 µl can be used without affecting the protocol

Conversion time

Large quantities of DNA may require longer conversion time to achieve complete conversion. For quantities of DNA greater than 1 µg, or if using DNA that is fragmented, werecommend performing a 5 hour incubation at 50°C to efficiently convert DNA samples.
We do not recommend using less than 500 ng of input DNA if the sample material isfragmented as this will lead to reduced DNA recovery. For DNA sequences that are GC-richand/or contain extensive secondary structures we suggest a 9 hour conversion at 50°C.
Longer conversion times, up to 16 hours, may be used if noticing incomplete conversion,
however, this may result in degradation of the DNA. If noticing a loss of DNA after theconversion reaction, the conversion time can be reduced to 3 hours at 50°C.

No PCR product

Conversion reagent is sensitive to air and moisture. If conversion reagent was not properly
sealed and stored, repeat conversion reaction using a new tube of conversion reagent.
Test primers on control bisulfite converted DNA. Evaluate the optimal PCR conditions
including annealing temperature, primer and magnesium concentrations. Consider designing new primers.
We recommend performing a Hot Start PCR reaction using a polymerase that has beenvalidated to work with bisulfite converted DNA. Follow the recommendations of the
manufacturer for the preparation of the PCR reaction.
If working with low amounts of Input DNA, such as 200 pg, it may be difficult to PCR
amplify the bisulfite converted DNA for visualization on an agarose gel. Concentrate DNA
prior to PCR to use more DNA per reaction, or use a different analysis method.

Can I store my conversion The DNA is recommended to be stored at 4°C for 5 days. If longer storage is needed, store
reaction samples forsamples at -20°C. As precipitates can form during -20°C, long term storage is not recomlonger than 5 days before
mended.
performing the DNA
purification and desulfonation steps?
Storage of converted DNA

Once DNA is prepared using Bisulfite Conversion Kit, samples may be stored at -20°C prior
to PCR analysis

How do I quantify mybisulfite converted DNA?

Bisulfite converted DNA will resemble RNA as it is single stranded and contains uracil inplace of the unmethylated cytosines. Use a spectrophotometer set to measure RNA witha conversion factor of 40 µg/ml for A260nm = 1.0. However, if the bisulfite converted DNA
has been PCR amplified prior to quantification, traditional methods to quantify DNA canbe used.

www.activemotif.com

18Section K. Related Products
DNA Methylation		Bisulfite Conversion Kit		
MethylCollector™ Ultra		
HypoMethylCollector™		
MeDIP		Hydroxymethyl Collector™		
hMeDIP		
PvuRts1I restriction enzyme		
b-Glucosyltransferase enzyme		
Recombinant TET1 protein, active		
DNMT Activity / Inhibition Assay		
Methylated DNA Standard Kit		
5-Carboxylcytosine DNA Standard		
Fully Methylated Jurkat DNA		
Jurkat genomic DNA		

Format50 rxns
30 rxns
30 rxns
10 rxns
25 rxns
10 rxns
50 units
500 units
25 µg
1 x 96 rxns
3 x 2.5 µg
0.5 µg
10 µg
10 µg

Catalog No.
55016
55005
55004
55009
55013
55010
55011
55012
31363
55006
55008
55014
55003
55007

Antibodies

FormatCatalog No.

100 µg100 µl100 µl100 µg100 µl50 µg
100 µg200 µl100 µg100 µg100 µg100 µg100 µl200 µg100 µg200 µl100 µg100 µg100 µg100 µg100 µl100 µg100 µl100 µg200 µ1

61111
61225
61223
39999
39769
39649
61255
39203
39204
39205
39206
39207
39907
39365
39215
39547
39216
39217
39218
61291
61443
61389
61395
39741
39625

Application

3-Methylcytosine rabbit pAb
DB
5-Carboxylcytosine rabbit pAb
DB, IF
5-Formylcytosine rabbit pAb
DB, IF
5-Hydroxymethylcytidine mouse mAb
DB, MeDIP
5-Hydroxymethylcytidine rabbit pAb DB, IF, IHC, MeDIP
5-Methylcytosine mouse mAb
DB, FACS, IHC, IP, MeDIP
5-Methylcytosine rabbit pAb
DB, IP, MeDIPCGBP rabbit pAb
WB
DNMT1 mouse mAb
ChIP, IHC, IP, WB
DNMT2 rabbit pAb
WB
DNMT3A mouse mAb
ChIP, IF, IHC, WB
DNMT3B mouse mAb
ChIP, IF, IP, WB
DNMT3L rabbit pAb
WB
Kaiso mouse mAb
WB
MBD1 mouse mAb
WB
MBD2 rabbit pAb
WB
MBD3 mouse mAb
WB
MBD4 rabbit pAb
WB
MeCP2 rabbit pAb
WB
MeCP2 mouse mAb
ChIP, IF, IHC, IP, WB
Tet1 rabbit pAb
ChIP, WB
Tet2 mouse mAb
IP, WB
Tet3 rabbit pAb
WB
Ubiquitin mouse mAb
IF, IHC, IP, WB
Uhrf1 rabbit pAb
WB

For more information or to see a complete list of DNA methylation antibodies, please visit our
website at www.activemotif.com/methylabs.

www.activemotif.com

19Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USA
Toll Free:877 222 9543
Telephone: 760 431 1263
Fax:
760 431 1351
E-mail:tech_service@activemotif.com
Active Motif Europe
Avenue Reine Astrid, 92
B-1310 La Hulpe, Belgium
UK Free Phone: 0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone:		+32 (0)2 653 0001
Fax:		
+32 (0)2 653 0050
E-mail: 		
eurotech@activemotif.com
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
Fax:
+81 3 5261 8733
E-mail:japantech@activemotif.com
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20