Instruction Manual

Premium Bisulfite kit
Cat. No. C02030030

Version 1 / 18.09.13

For Research Use Only.
Not intended for any animal or human therapeutic or diagnostic use.
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ContentProduct Contents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4Introduction to DNA Methylation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  5Product Description. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6Product Specifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  6Reagent Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7Protocol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  8Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  9FAQ's . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  10Ordering Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  Back Coverwww.diagenode.com |PAGE 4

DIAGENODEPREMIUM BISULFITE Kit | MANUALMANUALIntroduction• Fastest method for complete bisulfite conversion of DNA for methylation analysis.
• Ready-to-use conversion reagent is added directly to DNA.
• High-yield, converted DNA is ideal for PCR, MSP, array, bisulfite and Next-Gen Sequencing.

Product Contents
Description

Quantity

Storage

Conversion Reagent*

5 tubesRoom Temperature

Binding buffer

30 ml

Room Temperature

Wash buffer**

6 ml

Room Temperature

Desulphonation buffer

10 mlRoom Temperature

Elution buffer

1 mlRoom Temperature

Spin columns

50 columns

Room Temperature

Collection tubes

50 tubes

Room Temperature

Instruction Manual

1-Note - Integrity of kit components is guaranteed for one year from date of purchase. Reagents are routinely tested ona lot-to-lot basis to ensure they provide maximal performance and reliability.
* The Conversion reagent is in a ready-to-use liquid format. The reagent should be stored tightly capped at roomtemperature with minimum exposure to light.
** A dd 24 ml of 100% ethanol to the 6 ml Wash buffer concentrate (K07291001) before use.

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PREMIUM BISULFITE Kit | MANUALDIAGENODE

Introduction to DNA Methylation
Cytosine methylation is a naturally occurring base modification, in both prokaryotic and eukaryotic organisms,
consisting of the addition of a methyl group to the fifth carbon position of the cytosine pyrimidine ring via amethyltransferase enzyme (1). In prokaryotes DNA methylation provides a way to protect host DNA from digestion
by restriction endonucleases that are designed to eliminate foreign DNA. DNA methylation in higher eukaryotes
functions in the regulation/control of gene expression (2).
The majority of DNA methylation in mammals occurs in 5′-CpG-3′ dinucleotides, although other patterns doexist. About 80 percent of all 5′-CpG-3′ dinucleotides in mammalian genomes are found to be methylated, and themajority of the twenty percent that remain unmethylated are within promoters or in the first exons of genes. It has
been demonstrated that aberrant DNA methylation is a widespread phenomenon in cancer and may be among theearliest changes to occur during oncogenesis (3). DNA methylation has also been shown to play a central role in gene
imprinting, embryonic development, X-chromosome gene silencing, and cell cycle regulation.
The ability to detect and quantify DNA methylation efficiently and accurately has become essential for the study ofcancer, gene expression, genetic diseases, and many other important aspects of biology. To date, a number of methods
have been developed to detect/quantify DNA methylation including: high-performance capillary electrophoresis (4)and methylation-sensitive arbitrarily primed PCR (5). However, the most common techniques used today still rely on
bisulfite conversion (6).
Treating DNA with bisulfite chemically modifies non-methylated cytosines into uracil, methylated cytosines remainunchanged. Once converted, the methylation profile of the DNA can be determined using the desired downstream
application. For single locus analysis, the region of interest is generally amplified following bisulfite conversion (i.e.,bisulfite PCR) and then sequenced or processed for Pyrosequencing®. Recent advances in methylation detection
also allow the investigation of genome-wide methylation patterns using technologies including array-based methods,
reduced representation bisulfite sequencing (RRBS), and whole genome bisulfite sequencing (7).
Figure 2: DNA sequencing results following bisulfite treatment.
DNA with methylated C at nucleotide position #5 was processed using
the Premium Bisulfite kit. The recovered DNA was amplified by PCR
and then sequenced directly. The methylated cytosine at position #5
remains intact while the unmethylated cytosines at positions #7, 9,
11, 14 and 15 are completely converted into uracil following bisulfitetreatment (detected as thymine following PCR).

References
1. Adams RL. Bioessays. 1995; 17(2): 139-145.
2. Costello JF, Plass CJ. Med. Genet. 2001; 38(5): 285-303.
3. Stirzaker C. Cancer Res. 1997; 57(11): 2229-2237.
4. Fraga MF, et al. Electrophoresis. 2000; 21(14): 2990-2994.
5. Gonzalgo ML. Cancer Res. 1997; 57(4): 594-599.
6. Frommer M. Proc. Natl. Acad. Sci. USA. 1992; 89(5): 1827-1831.
7. Rakyan VK, et al. Nat. Rev. 2011, 12(8): 529-541.

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DIAGENODEPREMIUM BISULFITE Kit | MANUALMANUAL

Product DescriptionThe Premium Bisulfite kit features rapid and reliable bisulfite treatment and conversion of DNA for methylation
analysis. Key to the fast workflow is the ready-to-use Conversion reagent. No preparation is necessary, simply add thisunique reagent to a DNA sample, wait about an hour, and let the reaction proceed to completion. DNA denaturation
and bisulfite conversion processes are combined with added heat to facilitate rapid denaturation. Desulphonation andclean-up of the converted DNA is performed using a unique low-elution spin column. High yield, converted DNA isideal for PCR, array, bisulfite and Next-Generation Sequencing, etc.
Figure 2: Methylation Plot From Reduced Representation
Bisulfite Sequencing (RRBS).
Data shows the relative percentage of methylation at
individual CpG sites in mouse DNA. Methylation percentage
is shown across a ~3 Mb region of mouse chromosome
19. Bisulfite sequencing libraries were prepared using
mouse genomic DNA prepped with the Genomic Clean& Concentrator™ (D4010, D4011 – Zymo Research) andbisulfite converted using Premium Bisulfite kit technology
prior to Next-Gen Sequencing.

Figure 3: Spin column Design Characteristics.).
The image above shows the unique design of the column thatfacilitates extremely small elution volumes (≥10 μl) without
buffer carryover. This is unlike other columns that can retainliquid (binding/wash buffer residue) leading to carryover intothe eluate.

Specifications• DNA Input: Samples containing between 100 pg to 2 μg of DNA. For optimal results, the amount of input DNA
should be from 200 to 500 ng.
• Conversion Efficiency: > 99.5% of non-methylated C residues are converted to U; > 99.5% protection ofmethylated cytosines.
• DNA Recovery: >80%

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PREMIUM BISULFITE Kit | MANUALDIAGENODE

Reagent Preparation• Preparation of Wash buffer
Add 24 ml of 100% ethanol to the 6 ml Wash buffer concentrate (K07291001) before use.

Figure 4: Overview of Bisulfite Conversion.
Steps 1 and 2 occur during bisulfite conversion, while Step 3
is performed as the DNA is bound to the column matrix. For
the reaction to proceed to completion, it is essential the DNA
be fully denatured.

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PREMIUM BISULFITE Kit | MANUALDIAGENODE

MANUAL

Protocol1.

Add 130 μl of Conversion reagent to 20 μl of a DNA sample in a PCR tube. Mix, then centrifuge briefly to ensure
there are no droplets in the cap or sides of the tube.
Note: If the volume of DNA is less than 20 μl, compensate with water.
Samples >20 μl must be processed using multiple conversion reactions. Replicate reactions can becleaned using the same column for each by repeating steps 3-5.

2.

Place the PCR tube in a thermal cycler and perform the following steps:
a. 98°C for 8 minutes
b. 54°C for 60 minutes
c. 4°C storage for up to 20 hours
Note: The 4°C storage step is optional.

3.

Add 600 μl of Binding buffer to a Spin column and place the column into a provided Collection tube.

4.

Load the sample (from Step 2) into the Spin column containing the Binding buffer. Close the cap and mix byinverting the column several times.

5.

Centrifuge at full speed (> 10,000 x g) for 30 seconds. Discard the flow-through.
The capacity of the collection tube with the column inserted is 800 μl. Empty the collection tube whenevernecessary to prevent contamination of the column contents by the flow-through.

6.

Add 100 μl of Wash buffer to the column. Centrifuge at full speed for 30 seconds.

7.

Add 200 μl of Desulphonation buffer to the column and let stand at room temperature (20-30°C) for 15-20minutes. After the incubation, centrifuge at full speed for 30 seconds.

8.

Add 200 μl of Wash buffer to the column. Centrifuge at full speed for 30 seconds. Repeat this wash step.

9.

Place the column into a 1.5 ml microcentrifuge tube and add 10 μl of Elution buffer directly to the column
matrix. Centrifuge for 30 seconds at full speed to elute the DNA.
Alternatively, water or TE (pH ≥ 6.0) can be used for elution if required for your experiments.
The DNA is ready for immediate analysis or can be stored at or below -20°C for later use. For long term
storage, store at or below -70°C. We recommend using 1-4 μl of eluted DNA for each PCR, however, up to
10 μl can be used if necessary. The elution volume can be > 10 μl depending on the requirements of yourexperiments, but small elution volumes will yield higher DNA concentrations.

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PREMIUM BISULFITE Kit | MANUALDIAGENODEAppendix: Bisulfite Conversion and PCR Optimization
1.

Bisulfite Conversion of Double Stranded DNA Templates. The following illustrates what occurs to a DNA
template during bisulfite conversion.
Template: 		

A: 5’-GACCGTTCCAGGTCCAGCAGTGCGCT-3’				

B: 3’-CTGGCAAGGTCCAGGTCGTCACGCGA-5’

Bisulfite Converted:

A: 5’-GATCGTTTTAGGTTTAGTAGTGCGTT-3’				

B: 3’-TTGGCAAGGTTTAGGTTGTTATGCGA-5’

Note: Methylated “C” is underlined in the examples.
Note: Following bisulfite conversion, the strands are no longer complementary.
2.

PCR Primer Design. Generally, primers 26 to 32 bases are required for amplification of bisulfite converted
DNA. In general, all Cs should be treated as Ts for primer design purposes, unless they are in a CpG context.
See example below.
Bisulfite Converted:

A: 5’-GATCGTTTTAGGTTTAGTAGTGCGTT-3’

Primers: Reverse: 			
Forward: 		

3’-ATCATCACRCAA-5’ 		

R= G/A

5’-GATYGTTTTAGGT-3’ 				Y= C/T

Note: Only one strand (A) is amplified by a given primer set. Only the reverse primer binds to the converted
DNA, the forward primer will bind the strand generated by the reverse primer.
If the primer contains CpG dinucleotides with uncertain methylation status, then mixed bases with C and T (orG and A) can be used. Usually, there should be no more than one mixed position per primer and it should belocated toward the 5’ end of the primer. It is not recommended to have mixed bases located at the 3’ end of theprimer.
3.

Amount of DNA Required for Bisulfite Conversion. The minimal amount of human or mouse genomic DNA
required for bisulfite treatment and subsequent PCR amplification is 100 pg. The optimal amount of DNA perbisulfite treatment is 200 to 500 ng. Although, up to 2 μg of DNA can be processed, it should be noted that highinput levels of DNA may result in incomplete bisulfite conversion for some GC-rich regions.

4.

PCR Conditions. Usually, 35 to 40 cycles are required for successful PCR amplification of bisulfite converted
DNA. Optimal amplicon size should be between 150-300 bp; however larger amplicons (up to 1 kb) can begenerated by optimizing the PCR conditions. Annealing temperatures between 55-60°C typically work well.
As most non-methylated cytosine residues are converted into uracil, the bisulfite-treated DNA usually isAT-rich and has low GC composition. Non-specific PCR amplification is relatively common with bisulfite
treated DNA due to its AT-rich nature. PCR using “hot start” polymerases is strongly recommended for theamplification of bisulfite-treated DNA.

5.

Quantifying Bisulfite Treated DNA. Following bisulfite treatment of genomic DNA, the original base-pairing nolonger exists since non-methylated cytosine residues are converted into uracil. Recovered DNA is typically A, U,and T-rich and is single stranded with limited non-specific base-pairing at room temperature. The absorption
coefficient at 260 nm resembles that of RNA. Use a value of 40 μg/ml for Ab260 = 1.0 when determining theconcentration of the recovered bisulfite-treated DNA.

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DIAGENODEPREMIUM BISULFITE Kit | MANUALMANUAL

Frequently Asked Questions
Q: Should the input DNA be dissolved in TE, water, or some other buffer prior to its conversion?
A: Water, TE or modified TE buffers can be used to dissolve the DNA and do not interfere with the conversion
process.
Q: Which Taq polymerase(s) do you recommend for PCR amplification of converted DNA?
A: We recommend Diagenode's MethylTaq DNA polymerase (Cat. No. C09010010).

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PREMIUM BISULFITE Kit | MANUALDIAGENODE

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