Analysis of DNA Methylation in Plants by Bisulfite Sequencing

Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 631)

Abstract

Methylation of cytosines is a very important epigenetic modification of genomic DNA in many different eukaryotes, and it is frequently involved in transcriptional regulation of genes. In plants, DNA methylation is regulated by a complex interplay between several methylating and demethylating enzymes. Analysis of the resulting cytosine methylation patterns with the highest resolution is achieved after sodium bisulfite treatment, deaminating nonmethylated cytosines to uracil. Subsequent PCR and sequence analysis of individual amplicons displays the degree, position, and sequence context of methylation of every cytosine residue in individual genomic sequences. We describe the application of bisulfite sequencing for the analysis of DNA methylation at defined individual sequences of plant genomic DNA.

Key words

DNA methylation 5-methylcytosine (5mC) Bisulfite sequencing Bisulfite primer design Bisulfite conversion control 

1 Introduction

Methylation at position 5 of cytosines is a major epigenetic modification in eukaryotes and the only known covalent change of plant genomic DNA itself. Alterations in DNA methylation are frequently involved in transcriptional gene regulation (1, 2). Therefore, there is a great interest in analyzing cytosine methylation levels and distribution within the genome. Methylated and unmethylated cytosines can be distinguished by bisulfite genomic sequencing at single-base resolution. Treating genomic DNA with sodium bisulfite converts unmethylated cytosine to uracil, while 5-methylcytosine remains unchanged (Fig. 1). Amplification by the polymerase chain reaction (PCR) of converted DNA followed by sequencing reveals positions of 5-methylcytosine in the sequence of interest. This principle, first described by Frommer et al. (3) and Clark et al. (4), has since undergone several experimental simplifications and refinements and is widely applied to DNA from many different organisms. It is also applied for genome-wide analysis of DNA methylation (5-7).
Fig. 1.

Bisulfite conversion. DNA is denatured and then treated with sodium bisulfite, causing deamination of unmethylated cytosine to uracil which is converted to thymine by PCR

Here, we describe a simple and reliable protocol for DNA methylation detection by bisulfite sequencing of a specific target sequence. To succeed in generating meaningful data, complete conversion of unmethylated cytosines is the most important step. This is achieved by incubating genomic DNA in a high bisulfite concentration at high temperature and low pH. The conversion procedure and subsequent purification lead to DNA fragmentation and DNA loss, respectively, requiring a balance between conversion efficiency and DNA stability. Based on our experience and in the interest of reproducible experiments, we recommend commercially available kits for the conversion procedure, and here we will focus on crucial pre- and postconversion steps. Among these are the DNA preparation, conversion control, the design of bisulfite primers, and cloning of amplified sequences. Tools for data analysis and comments on interpretation are described in the chapter “Analysis of bisulfite sequencing data from plant DNA using CyMATE.”

2 Materials

2.1 Extraction and Pretreatment of Genomic DNA

  1. 1.

    Nucleon PhytoPure (Amersham Biosciences) (see Note 1).

     
  2. 2.

    RNase A, DNase- and protease-free (10 mg/ml, Fermentas) (see Note 2).

     
  3. 3.

    Restriction endonuclease and appropriate buffer (see Note 3).

     
  4. 4.

    3 M sodium acetate.

     
  5. 5.

    Ethanol - absolute and 70%.

     

2.2 Sodium Bisulfite Conversion and PCR Amplification

  1. 1.

    EpiTect Bisulfite Kit (Qiagen) (see Note 4).

     
  2. 2.

    Primer for conversion control (see Note 5).

     
  3. 3.

    Primer for the region under investigation (see Note 6).

     
  4. 4.

    TrueStart Taq DNA Polymerase (Fermentas) (see Note 7).

     
  5. 5.

    dNTP set - 100 mM aqueous solutions at pH 7.0 of each of dATP, dCTP, dGTP and dTTP (Fermentas or equivalent product).

     
  6. 6.

    QIAquick Gel Extraction Kit (Qiagen or equivalent product).

     

2.3 Cloning and Sequencing of PCR Products

  1. 1.

    pGEM-T Easy Vector System (Promega or equivalent product).

     
  2. 2.

    Competent E. coli (DH5α).

     
  3. 3.

    LB solution containing 50 mg/ml Ampicillin.

     
  4. 4.

    LB plates containing 50 mg/ml Ampicillin, 0.5 mM IPTG and 80 µg/ml X-Gal.

     

3 Methods

3.1 Extraction and Pretreatment of Genomic DNA

  1. 1.

    Extract genomic DNA from the plant material under investigation according to the manufacturers’ instructions. To achieve optimal bisulfite conversion rates, genomic DNA needs to be clean and intact (see Note 1) and should be from young and healthy tissues. As an example, 100 mg of 3 week old Arabidopsis seedlings give good and reproducible results. Additional RNase treatment is recommended (see Note 2) and can be applied during cell lysis (30 min at 37°C). After the preparation, resuspend genomic DNA in 50 µl sterile water, heat it to 55°C for 30 min with constant slight shaking (600 rpm), and keep it on ice until usage. Alternatively, the dissolved DNA can be kept at 4°C overnight.

     
  2. 2.

    Measure the DNA concentration photometrically and check DNA integrity by gel electrophoresis of a 1 μl aliquot. The DNA should appear as a single band. Digest 2 µg genomic DNA with an appropriate restriction enzyme (see Note 3) (5–10 U/µg genomic DNA in the recommended buffer) and incubate overnight.

     
  3. 3.

    Precipitate the digested DNA with 1/10 volume of 3 M sodium acetate and three volumes of absolute ethanol (−20°C, >2 h). Centrifuge and remove the supernatant, and wash the pellet with 500 µl 70% ethanol. Dry the pellet and resuspend it in 20 µl sterile water. Keep the digested DNA at 4°C until usage but not more than a month. The sample is now ready for bisulfite conversion.

     

3.2 Sodium Bisulfite Conversion and PCR Amplification

  1. 1.

    One of the most important and critical issues for successful bisulfite sequencing is an accurate primer design. This is challenging because information about the degree of methylation, and thereby the expected sequence after conversion is an experimental question that is not available beforehand. To ensure unbiased results, cytosine residues at primer binding sites should be set to match degenerate bases in primers, but the number of degenerate positions should be kept small. Therefore, there are special constraints on the primers and their location on the DNA template. In addition, DNA strands need to be analyzed separately, since they are no longer complementary. With some experience, manual selection of bisulfite primer sets worked well for us, but there is a software primer design tool for bisulfite-converted plant genomic DNA (8), (for more details about bisulfite primer design, see Note 6).

     
  2. 2.

    As stated in the introduction (see Note 4), we recommend to apply a commercially available bisulfite sequencing kit to assure complete and reproducible conversion. Perform the procedure with the desired amount of DNA and according to the protocol supplied with the kit. We have improved the results by extending the conversion procedure for an extra 5 min at the denaturation step at 99°C, and by adding an additional 2 h conversion step at 60°C before a final hold step at 20°C. We use a PCR machine to control temperature and duration of denaturation and incubation times.

     
  3. 3.
    Check the completeness of conversion by PCR with primers matching a fully converted or a nonconverted site in a region known to be unmethylated (see Note 5 and Fig. 2).
    Fig. 2.

    The expected pattern after conversion control PCR, using two different primer sets distinguishing between unconverted DNA (BScontrol1) and converted DNA (BScontrol2)

    Typical PCR conditions for conversion control primer sets are:

    Reaction set-up (see Note 7):

    Sterile water

    11.3 µl

    10× TrueStart Taq buffer

    2.5 µl

    dNTP mix, 2 mM each

    2.5 µl

    Forward primer, 10 µM

    2.0 µl

    Reverse primer, 10 µM

    2.0 µl

    MgCl2, 25 mM

    1.5 µl

    TrueStart Taq DNA Polymerase

    0.2 µl

    Converted DNA

    3.0 µl

    Total volume

    25.0 µl

    Thermal cycling conditions:

    95°C

    2 min

    1 cycle

    95°C

    30 s

     

    50°C

    30 s

    35 cycles

    68°C

    30 s

     

    68°C

    2 min

    1 cycle

    Optional: To determine the degree of conversion efficiency, the amplicon from the conversion control PCR (primer set: BScontrol2F and BScontrolR) can be sequenced, or cloned and sequenced (see below). Only samples with high conversion rates (>95% of all C converted to T) should be used for amplification of the experimental region.

     
  4. 4.

    If the control indicates the full conversion of DNA, start PCR of the target region with bisulfite primers. We recommend using Hot Start Taq polymerase with a good performance (see Note 7).

    Typical PCR conditions for the experimental target primer sets (see Note 8):

    Reaction set up:

    Sterile water

    11.3 µl

    10× TrueStart Taq buffer

    2.5 µl

    dNTP mix, 2 mM each

    2.5 µl

    Forward primer, 10 µM

    2.0 µl

    Reverse primer, 10 µM

    2.0 µl

    MgCl2, 25 mM

    1.5 µl

    TrueStart Taq DNA Polymerase

    0.2 µl

    Converted DNA

    3.0 µl

    Total volume

    25.0 µl

    Thermal cycling conditions (see Note 8):

    95°C

    2 min

    1 cycle

    95°C

    30 s

     

    X°C

    30 s

    35–45 cycles

    68°C

    Y s

     

    68°C

    2 min

    1 cycle

     
  5. 5.

    Analyze an aliquot of 5 µl PCR reaction by electrophoresis on an agarose gel. If the amplicon is only a single sharp band of the expected size, the PCR product can be directly used for cloning. If the expected product is visible and accompanied by some unspecific bands or smear, we recommend gel extraction of the specific amplicon using an appropriate column, e.g., from the QIAquick Gel Extraction Kit (Qiagen), prior to cloning. If the band is not or hardly visible, load the whole PCR reaction and perform gel extraction (see Note 9).

     

3.3 Cloning and Sequencing of PCR Products

  1. 1.

    If there is a sufficient amount of converted DNA at the start of the PCR, the amplicon represents different copies of genomic DNA. Therefore, comparison of several individual plasmid clones obtained via PCR allows a statistical analysis. It also reveals the variability of methylation patterns among genomic copies. Efficient cloning can be obtained using T/A cloning systems that include blue/white screening for recombinant plasmids (see Note 10). Add 3 µl of the purified PCR product in a 10 µl ligation mix, and use the pGEM-T Easy Vector System for optimal results. Ligation is performed at 4°C overnight. Five µl of the ligation mix are used to transform competent E. coli. The transformed bacteria are plated on LB-Amp/IPTG/X-Gal agar plates and incubated overnight at 37°C.

     
  2. 2.

    White colonies (15–20) are picked from these plates and dipped briefly into a PCR tube containing colony-PCR mix prior to inoculation in 2 ml LB-Amp and incubation at 37°C.

    Colony PCR mix using standard PCR solutions (see Note 11):

    Reaction set up:

    Sterile water

    14.4 µl

    10× standard PCR buffer (containing MgCl2)

    2.0 µl

    dNTP mix, 2.5 mM each

    1.5 µl

    Primer M13 forward, 10 µM

    1.0 µl

    Primer M13 reverse, 10 µM

    1.0 µl

    Taq DNA Polymerase (5 U/µl)

    0.1 µl

    Total volume

    20.0 µl

    Thermal cycling conditions:

    95°C

    2 min

    1 cycle

    95°C

    30 s

     

    60°C

    30 s

    25–30 cycles

    72°C

    30 s

     

    72°C

    2 min

    1 cycle

     
  3. 3.

    Run the PCR products on agarose gels, identify those with the expected size, and prepare plasmid DNA from the corresponding liquid cultures. These plasmids can be controlled further by EcoRI digestion for the presence of the correct insert size or directly prepared for sequence analysis. We recommend analyzing at least ten independent clones per amplicon by sequencing. Sequencing is done by standard methods, priming with M13 forward or M13 reverse primers (see Note 12).

     
  4. 4.

    The sequence analysis of individual clones provides useful information about the degree of methylation in each clone and of each cytosine residue as well as the sequence context of methylated cytosine. The alignment of individual sequence files is not trivial due to different reading starts and sequence heterogeneity after conversion. A manual comparison of sequences can be facilitated by aligning the whole set of sequence files and creating a blunt-ended multiple sequence alignment, excluding primer and vector sequences (see Note 13). There are several publicly available software tools supporting the analysis of bisulfite data using algorithms considering the plant-specific diversity of DNA methylation (8-10).

     

4 Notes

  1. 1.

    Bisulfite sequencing requires clean and high molecular weight genomic DNA. In our hands, DNA preparations using standard lab protocols, even if they are sufficient for routine PCR, have not been successful for good bisulfite conversion. However, the positive experience with PhytoPure does not exclude using other procedures from other suppliers that provide good quality genomic DNA. This is also true for all other recommended chemicals, enzymes and kits.

     
  2. 2.

    RNase A treatment should always be performed during or subsequent to genomic DNA extraction. If conversion problems occur, we recommend an additional Proteinase K treatment of genomic DNA upon extraction to eliminate possible protein contamination.

     
  3. 3.

    Cleavage of genomic DNA by restriction endonucleases is recommended prior to conversion to ensure the release of secondary structures and allow for full denaturation. This is important, as sodium bisulfite can only react with cytosine residues in single-stranded DNA. The enzyme should be chosen to yield sufficiently large fragments (a six-base recognition site) and not to be inhibited by DNA methylation. For example, suitable enzymes are BamHI, DraI or SspI, which all give an approximate fragment size of 4 kb. Care has to be taken not to cut in the sequence between bisulfite primers.

     
  4. 4.

    Most commercially available bisulfite conversion kits are applicable to a very small amount of DNA and guarantee reproducible conversion rates and DNA integrity. They support DNA denaturation and include DNA protection buffers to prevent DNA fragmentation due to depurination caused by harsh conversion conditions. They also simplify and improve tedious purification procedures after conversion, and speed up the analysis. The following bisulfite conversion kits, in alphabetical order of supplier and without any claim for completeness, function equally good: MethylDetector Bisulfite Modification Kit (Active Motif), Methyl SEQr Bisulfite Conversion Kit (Applied Biosystems), Methyl Easy Kit (Diagenode), MethylCode Bisulfite Conversion Kit (Invitrogen), EZ DNA Methylation Kit (Zymo Research).

     
  5. 5.

    A decisive step for the analysis is the degree of conversion of unmethylated cytosines in a DNA sample. If unmethylated cytosines are not completely modified, all the following results are meaningless. There are different options to assure completeness of the reaction. A genomic DNA sample can be spiked with unmethylated DNA (3), e.g., with plasmid DNA amplified in an appropriate bacterial strain or by PCR. Alternatively, bisulfite-converted DNA is analyzed by two PCR reactions using primers that match either the fully converted or nonconverted sequence in the same C-rich region with a genomic target region known to be unmethylated (7, 9). We recommend the latter option, as this control is more accurate and closer to the conditions of the experimental region. As an example, for the analysis of Arabidopsis DNA, the following upstream primers specific for either nonconverted or converted DNA (At5g66750) have yielded good results (Fig. 2) in combination with the same downstream primer in a C-free region.

    BScontrol1F (nonconverted DNA):

    5′CGTCTGGTGATTCACCCACTTCTGTTCTCAACG-3′BScontrol2F (converted DNA):

    5′-TGTTTGGTGATTTATTTATTTTTGTTTTTAATG-3′BScontrolR (unbiased):

    5′-CTCTCACTTTCTATCCCATTCTA-3′

     
  6. 6.

    The upper limit of PCR products derived from bisulfite-treated DNA should be not more than 500 bp. We have obtained the best results with primer sets generating amplicons of 200–300 bp. Primers should contain degenerated nucleotides (R for A/G in one primer, Y for C/T in the other primer) to allow unbiased amplification of methylated and unmethylated DNA. Not more than two to three degene­rated sites should be present in one primer (see below). Try to find regions relatively rich in Gs and poor in Cs on the strand you are interested in. Due to conversion, the top and bottom strand are no longer complementary, which may lead to strand-specific amplification. The 3′ primer complementary to the sequenced strand should contain degenerated nucleotides for A/G (R), as it should potentially bind to uracil converted from unmethylated cytosines and to unchanged methylated cytosines in the first round of amplification. The 5′ primer homologous to the sequenced strand should contain Y standing for C/T to anneal to the already amplified strand generated with the 3′ primer. Repeats of dinucleotides (e.g., ATATATAT) or primers with long runs (>4 b) of a single base should generally be avoided, as they can misprime. Similar to other standard PCR reactions, primer design should avoid regions of homology outside of the target. Therefore, we recommend running a BLAST search with designed primers, if genome information for an organism is available. In addition, primers should be designed to avoid secondary structure and primer dimer formation. A primer length can be varied to adjust annealing temperatures, since primers from 21 to 28 nucleotides worked well in our hands. The melting temperature can range between 48 and 60°C, but primer pairs should have no more than 4°C difference in melting temperatures. We recommend running an initial gradient PCR with any new bisulfite primer set to find the most appropriate annealing temperature. If the amount of bisulfite-treated samples available allows using converted DNA for primer testing, we absolutely advise to do so.

     
  7. 7.

    The use of hot-start polymerase from this or other suppliers is recommended to avoid nonspecific primer amplification.

     
  8. 8.

    Reaction conditions like the annealing temperature X and the extension time Y need to be adjusted for each amplicon and primer set depending on the melting temperature, distance and base composition (see also Note 6). If one primer contains more degenerated sites than the other, we recommend an adjustment of primer concentration in the PCR setup, i.e., a higher amount of primer with the higher number of wobble bases. The equilibrium between molar ratios for each primer pair is very important, as the formation of hybrid PCR products can occur during PCR. An incomplete extension product is able to act as a highly efficient primer in a subsequent PCR cycle, resulting in the formation of a hybrid product containing information from the bottom and top strand (11).

     
  9. 9.

    We use 1.5% agarose gels, but their concentration should be adjusted to the size of the PCR product. In case of low efficiency of bisulfite PCR, it is advisable to load the entire PCR reaction and gel purifying a band. Column purification of gel-extracted PCR products is very efficient. A sample should not be diluted too much; elution in 20–30 µl sterile water from the column works best. The elution step should be repeated with the first eluate. We do not recommend any nested PCR approaches, since they can cause some bias toward either methylated or unmethylated targets and may increase redundancy rather than enhancing sensitivity or specificity.

     
  10. 10.

    We compared blunt end cloning and T/A cloning systems. T/A cloning was much more efficient and gave more transformants with an expected insert. Although ligation should be unbiased with regard to direction, we observed a preferential insert orientation of the cloned fragment, which could be due to a particular sequence composition of bisulfite-converted DNA.

     
  11. 11.
    Sequence for colony PCR primers:

    M13 forward

    5′ GTA AAA CGA CGG CCA G 3′

    M13 reverse

    5′ CAG GAA ACA GCT ATG AC 3′

     
  12. 12.

    The amplified converted target sequence often results in repetitive and A/T rich sequences. Some sequencing systems may have problems to produce sequencing runs of sufficient length and quality. In our hands, the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) is very useful.

     
  13. 13.

    For significantly methylated genomic regions, patterns of individual clones are usually so diverse that clones with identical sequences indicate PCR-generated redundancy rather than identical genomic templates. It is advisable to exclude duplicates from quantitative analysis.

     

References

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Andrea M. Foerster
    • 1
  • Ortrun Mittelsten Scheid
    • 1
  1. 1.Gregor Mendel Institute of Molecular Plant BiologyAustrian Academy of SciencesViennaAustria

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