Schumacher’s guide for:

 

Bisulfite conversion of DNA for methylation fine-mapping.

 

Dr. Axel Schumacher; xdr.a.schumacher@gmail.comx

Version 1.b; Last modified 25th of January 2007

 

 

1. Introduction

 

The method described in detail below is based on traditional bisulfite modifications, however several new improvements and many comments are included. This protocol requires at most only 1.5 hour bisulfite reaction. In this method, sodium bisulphite is used to convert cytosine residues to uracil residues in single-stranded DNA, under conditions whereby 5-methylcytosine remains non-reactive (Fig. 1). The converted DNA is then amplified with specific primers (where uracil corresponds to thymine in its base pairing behaviour), followed by downstream detection techniques, such as sequencing (with or without cloning) or microarrays. All the cytosine residues remaining in the interrogated sequence represent methylated cytosines in the genome.

This protocol can also be applied for small sample sizes of a few nanograms or below. The standard protocol uses 500 ng. The eluted DNA is suited for all techniques currently used for the analysis of DNA methylation; including PCR, real-time PCR, MSP-PCR, bisulfite sequencing, COBRA, microarrays and pyrosequencing.

 

 

Fig. 1: Step 1: Sulphonation; Step 2: hydrolytic deamination and Step 3: alkali-desulphonation. Bisulfite conversion is performed under acidic conditions and preferentially deaminates cytosine in a nucleophilic attack whilst the methyl group on 5-methylcytosine is protecting the amino group from the deamination.

 

Advantages of this protocol: Under known standard conditions at least 80% (up to ~98%) of the DNA is usually degraded (due to non-specific degradation under acidic conditions), which poses severe problems for subsequent analyses. Hence, this protocol was developed to improved several critical steps, which leads to higher recovery rates, a higher conversion rate (99.5-100%) and a much faster procedure. This method uses the presence of additional denaturing reagents and scavengers.

 

 

 

 

 

2. Materials

 

2.1 Required chemicals:

 

Chemical

Company

Sodium bisulfite (mixture of NaHSO3 and Na2S2O5)

Sigma, # 243973

Hydroquinone

Sigma, #H9003

NaOH pellets

Sigma

Guanidine Hydrochloride

Fluka #50937 (25 ml)

6-Hydroxy-2,5,7,8-tetramethylchroman-2-carbonsäure (called Trolox)

Sigma-Aldrich, #23,881-3 (1 g)

Tetraethylammoniumchloride (TAC)

Sigma, #T2265 (25 g)

Tetraethylenepentaminepenta-hydrochloride

(Also known as TETRAEN)

Sigma-Aldrich, #375683 (10 g)

Degassed water

 

 

 

2.2 Preparation of the solutions:

Note: Although it is not mentioned in most bisulfite protocols, it is highly recommend to degas the water since the oxygen (O2) in the water reacts with the bisulfite, reducing the bisulfite conversion rate significantly. The oxygen slips between the loose hydrogen-bonded network of water molecules without forcing them apart. The oxygen is then caged by water molecules, which weakly pin it in place. For example, at room temperature, about 8.24 mg oxygen is dissolved in every litre of water.

Note A: This unsaturated di-keton is an alkalizing antioxidant and forms the energetically similar p-benzochinone, thereby working as inhibitor of the unwanted bisulfite-oxidation reaction. Additionally, it prevents DNA strand breakage that can occur through depurination.

Note B: Instead of Hydrochinone it is also possible to use quinol (dihydroxy benzene), which is a white crystalline substance, C6H4(OH)2 and is obtained by the reduction of quinone. It is a diacid phenol, resembling, and metameric with, pyrocatechin and resorcin.

Note: TAC is a quarternary ammonium compound used as a catalyst of the conversion reaction. Other quaternary amine compounds (i.e quaternary alkyl ammonium salts) include quaternary alkyl ammonium chlorides such as quaternary methyl ammonium bromide, quaternary alkyl ammonium bromides, quaternary ammonium chlorides, tetraethyl ammonium hydroxide, tetraethyl ammonium chloride, tetrabutyl ammonium chloride, tetrabutyl ammonium bromide, among others.

 

 TAC

 

Note: After using the chemicals, if possible, sodium bisulfite and hydroquinone powders should be stored under vacuum or non-reactive nitrogen.

 

3. Method

 

3.1 Preparation of the bisulfite mix

  Sodium Bisulfite

 

Note A: Some protocols are carried out in the presence of ~25% Dioxan, one of its derivatives or a similar aliphatic cyclic ether. The bisulfite reaction can also be carried out in the presence of a n-alkylene glycol compound, particularly in the presence of their dialkyl ethers, and especially in the presence of diethylene glycol dimethyl ether (DME). However, in my experience, the introduction of additional solvents complicate the procedure especially since the salts in the bisulfite reaction will tend to precipitate out of solution, which in turn will lower the efficiency and will produce problems during the purification steps.

Note B: Alternatively to sodium bisulfite, 2.56 M sodium metabisulfite (e.g. Sigma S9000) can be used instead, which is equivalent to 5 M sodium bisulfite in molarity. As a solid, this product is sodium metabisulfite, Na2S2O5. Dissolved in water, it yields NaHSO3.

Note C: It is also possible to perform the bisulfite conversion using other bisulfite compounds, for example magnesium bisulfite, in a concentration from about 1-2 M. However, it is my experience that magnesium bisulfite (which comes already in solution), complicates the procedure since it will easily precipitate the additional components out of solution.

Note D: Usually and surprisingly unknown is that, instead of bisulfite, the conversion of cytosine to uracil can also be performed with a nucleophilic organo-sulfur compound (e.g of the structure X2-S(O)-X1, where X can be Me, Et or (CH2)5) ). Suitable mono-substituted sulfur nucleophiles are, for example, sulphurous acid monomethyl ester (e.g. monomethyl sulfite), methyl sodium sulfite, phenyl hydrogen sulfite, sodium phenyl sulfite, methylsulfinic acid or ethylsulfinic acid. Other possible substances comprise bis-substituted sulfur nucleophiles such as sulphurous acid dimethyl ester, methanesulfinylmethane, 2-methyl-propane-2sulfinic acid diethylamide, [1,3,2]Dioxathiolane 2-oxide or 2,5-Diethyl[1,2,5]thiadiazolidine 1-oxide, among others.

Note: Try to adjust the pH to 5.0.

Note: Trolox is an antioxidant and a water soluble analogue to vitamin E. This drug is known to penetrate rapidly the biological membranes and behaves as a powerful peroxyl radical scavenger. Following reconstitution, aliquot and freeze at –20°C or below. This product is stable for 3 years as supplied.

Note: It has been found that polyamines, such as the Tetraethylenepentaminepenta-hydrochloride, which I introduce here for this method, are useful catalysts in the bisulfite conversion of cytosine. The presence of a polyamine catalyst enables faster reaction times. The amine salt TETRAEN is the only known molecule with five catalytic amine groups. The amine groups harbour opposite charges which drive the electrons in the cytosine from the location at the pyrimidine ring where the bisulfite reaction takes place, thereby catalyzing the forward reaction. Polyamine catalysts work in a range of about 0.1 M. I tested several catalyst and had the best experiences with TETRAEN. Other polyamines (which are patented by others in combination with the bisulfite conversion) include diamines, triamines such as diethylene triamine (DETA; 2M), guanidine and tetramethyl guanidine; tetraamines, such as spermine and other compounds containing two or more amine groups, and their salts.

 

 TETRAEN

 

Note: Helps keeping the DNA single-stranded. It is known that Urea has similar properties and may enhance deamination. However, it was found by Hayatsu et al. 2006; that urea does not significantly improve the conversion reaction with bisulfite. Additionally, guanidine hydrochloride has similar properties as other polyamine catalysts (described above), therefore enhancing the conversion reaction.

 

Wrap this up in foil as well and heat at 55°C to help dissolve.

 

 

3.2 Preparation of the DNA

First of all, ensure that only high-quality DNA (preferentially phenol-chloroform extracted if applicable) is used for the conversion reaction. Other DNA sources will work too, but may be more difficult to analyze.

·        Split your DNA in four tubes.

·        For each tube, digest ca. 250 ng to 2 µg genomic DNA with a restriction enzyme outside the region of interest for 2h. A good combination is the following: Tube 1: EcoRI; Tube 2: PvuII; Tube 3: HindIII and Tube 4: No digest.

Note A: The purpose of digesting the DNA is to lower the viscosity and hence to facilitate a complete DNA denaturation by creating single stranded DNA molecules. Sodium bisulfite can only react with pyrimidines that are not involved in base-paring or for inaccessible 3D-structures. By using several different enzymes, only one BS preparation has to be performed, even if many different primer combinations will be used in downstream experiments. If only one enzyme would be used, the danger of having a restriction site within your PCR product would be too big.

Note B: This enzyme combination is most cost-efficient and reliable, i.e the fragment sizes are appropriate.

·        For the 4th tube, either sonicate the DNA briefly or press the DNA to a small syringe needle (e.g. a 19G or 21G syringe needle) to chop it to smaller pieces. Do not add any enzyme to this sample.

·        Heat-inactivate the restriction enzymes for at least 20 minutes according to the manufacturers guidelines.

·        Pool the DNA from all 4 tubes and mix. The DNA is now ready to be used for BS-conversion.

Note A: If enough DNA is present, I strongly recommend purifying/precipitating the DNA before proceeding to the BS reaction. Purification ensures that the DNA has a higher conversion rate (i.e. bound enzymes or remaining RNA molecules may interfere with the BS-conversion).

Note B: Some protocols use special matrices to attach the DNA during BS conversion (e.g. glass beads, agarose, silicon oxide, for example glass fleeces or fibers or magnetic glass particles among others). In the case of low-melting point agarose, the DNA is automatically kept in single-stranded conformation, therefore ensuring conversion. However, with the presented protocol it is not necessary to include a 3D-matrix, since the DNA will be kept single stranded in solution.

 

 

3.3 Bisulfite conversion

Note: It is not necessary to add NaOH to denature the DNA (but it also doesn’t hurt).

Note A: Since the bisulfite reaction is not overlaid with mineral oil and the volume is relatively large, a thermal-cycler with a heated lid should be used. It is also important to use are PCR tubes that really close tightly.

Note B: To ensure complete denaturation, no more than 2 µg of starting material should be used.

Note A (Temperature): Full cytosine deamination can be achieved under several time/temperature combinations. The sulphonation step itself is exothermic, however the overall deamination rate increases with raising temperature. I recommend a reaction temperature of 55 to 60°C, although higher temperatures are also possible. The risk with increasing the temperature is that also a higher degree of the DNA will degrade. Since this protocol is optimized for increased denaturation (remember: double-stranded DNA is non-reactive to bisulfite treatment), it seems not necessary to expose the bisulfite DNA to very high temperatures.

Note B (Conversion time): Since DNA degradation increases with time (other parameters: oxidative decay under acidic conditions, bisulfite concentration and reaction temperature) it is evident to shorten the incubation time as much as possible. In our lab we could produce PCR products from DNA that was bisulfite treated for merely 30 minutes. Yet, to ensure a complete conversion of all non-methylated cytosines, we usually increase the conversion time up to 1.5 hours, using a thermo-cycler. Longer incubation periods than 3 hours are not recommended since bisulfite will also deaminate methylcytosine if it has enough time.

Note C: The converted DNA should be processed as soon as possible, but in emergency, the converted DNA may be left in the thermal-cycler overnight without any significant loss of performance.

Note D: It is barely known that under certain conditions, also Thymine reacts with bisulfite, although with lower intensity, producing a dihydrothymine-6-sulfonate structure.

Note E: After bisulphite treatment, the strands are no longer complementary and strand specific primers can be designed. This allows amplification, cloning + sequencing of the individual strands (sense and antisense) to determine their methylation patterns. It can be assumed that methylation of both strands is identical (due to symmetric de novo methylation of a newly synthesized strand by the methyltransferase). Hence, usually there is no such need to sequence both strands. We sequenced many genomic regions to study this situation and could not find any significant difference between the strands for all interrogated genes.

 

 

3.4 Purification, Desulphonation and Neutralization

There are many ways of purifying the BS-DNA, but we will only cover one method that does not include the desulphonation step in a column, which are used by some kits. On one hand, to desulphonate during the purification has the benefit that the procedure is faster, on the other hand there is the risk that the desulphonation may be incomplete (e.g. due to the fact that the DNA may be inaccessible in the matrix or because it is difficult to heat the sample to the required temperature of 42°C) or that the column gets damaged which will lead to the loss of the BS-DNA sample. One has to keep in mind that the DNA has to be single stranded for the desulphonation, however in the denatured form it will likely slip through the silica matrix of a conventional purification column. A good purification is necessary to remove the bisulfite salt and the other chemicals used in the conversion process that may inhibit downstream procedures.

 

Purification (using the NucleoSpin Extract II kit)

Note: NucleoSpin columns work practically the same as the more widely used Qiagen columns but are much cheaper.

 

Desulphonation

Note A: Most protocols use 37°C; however, it was shown that an increase of the incubation temperature to 42°C is necessary to obtain full Denaturation/conversion of GC-rich DNA regions such as CpG islands.

Note B: This alkali treatment with NaOH  is necessary  to remove the bisulfite adduct from the uracil ring (uracil sulphonate). It is important that this desulphonation step is complete.

Note A: In case you work with low DNA concentrations (<200 ng), the addition of a carrier molecule (e.g. tRNA or Glycogen) in the precipitation is essential for the complete recovery of the bisulfite treated DNA, followed by two volumes Ethanol.

Note B: Also, in case of very low amounts of DNA, it is recommended to use Isopropanol instead of Ethanol for precipitation. Isopropanol has the advantage of precipitating DNA at lower concentrations. Instead of mixing two volumes of ethanol with the DNA-salt solution, addition of one volume of isopropanol will suffice.

Note C: Many older manuals indicate that precipitation should be allowed to occur in the cold (e.g. -20°C). This has been shown to be unnecessary, especially when using ammonium acetate.

Note D: Check the pH of your salt as it may affect the degree of precipitation significantly.

Note: A following BS-PCR will work even better if the BS-DNA is eluted in water. However, this will, of course, decrease the lifetime of the converted DNA dramatically.

 

3.5 Storage conditions of bisulfite-treated DNA. 

The DNA can be stored at –20°C. Different labs report very different times for how long the DNA can be used, from several days to up to 2 years. In my experience, bisulfite treated DNA can be stored for at least 1 month without affecting the PCR yield significantly, yet –the fresher, the better. I recommend to aliquot the BS-product into smaller samples which will be thawed only one by one when used. It may also be advisable to add the cyclic ether Tetrahydrofurfuryl alcohol (THFA, a solvent that is sometimes used in make up), to the storage/elution buffer, which helps partly to protect DNA degradation. THFA is a clear, colorless liquid that is used extensively in various industries as a high-purity, water miscible solvent, and as a chemical intermediate. (However, it is unknown to me what percentage of THFA is best. As a solvent it is often used with 2-20% of the total volume).

Note: Bisulfite treated DNA can not be easily quantified by spectrometry. BS-DNA is a mostly single stranded mixture of nucleic acids that has features of DNA and RNA. A good method to get some idea how much DNA is left is to run a few µl on an agarose gel. For more precise measurements, a HPLC analysis is required.

 

 Your Notes: