Microarrays and DNA methylation profiling

 

Dr. Axel Schumacher © 2004-2007

 

 

 

The method described in detail below is based on the analysis of the enriched unmethylated DNA fraction, using a series of treatments with methylation-sensitive restriction enzymes, adaptor ligation, PCR amplification, and quantitative mapping of unmethylated DNA sequences using microarrays. The key advantages of this approach are the ability to investigate DNA methylation patterns using very small DNA amounts and also relatively high informativeness in comparison to the other restriction enzyme- based strategies for DNA methylation profiling (1).

 

 

1. Introduction

 

The key principles and some technical details of this method are described in our recent article (1). Briefly, genomic DNA (gDNA) is digested with the methylation-sensitive restriction enzymes such as HpaII and Hin6I. Whereas methylated restriction sites remain unaltered, the sites containing unmethylated CpGs are cleaved by the enzymes, and DNA fragments with 5’-CpG protruding ends are generated. In the next step, the double-stranded adapter CG-1 is ligated to the CpG-overhangs. At this point, it is expected that most of the relatively short (<1.5 kb) and amplifiable DNA fragments derive from the unmethylated DNA regions. Some ligation fragments, however, may still contain methylated cytosines. A large proportion of these fragments are eliminated by treatment with McrBC, thereby increasing the specificity of the enrichment of the unmethylated DNA fraction. McrBC cleaves DNA containing methylcytosine on one or both strands, recognizing two half-sites of the form (G/A)mC; these half-sites can be separated by up to 3 kb, but the optimal separation is 55–103 base pairs. The remaining pool of unmethylated DNA fragments is then enriched by aminoallyl-PCR amplification that uses primers complementary to the adapter CG-1. An important advantage of using protruding ends in the adapter–ligation step is that degraded gDNA fragments will not be ligated and amplified and, therefore, will not interfere with DNA methylation analysis (which is especially useful when analyzing tissues with relatively long post-mortem interval or paraffin-embedded samples). The enriched unmethylated DNA fractions are then labelled with fluorescent dyes and hybridized to microarrays. Several different types of microarrays can be used for epigenetic analysis, for example oligonucleotide arrays of individual genes or microarrays containing relatively larger DNA fragments of gene regulatory regions, such as CpG islands (1).

 

 

Fig 1: Schematic outline of the microarray-based method for identification of DNA methylation differences in genomic DNA. Samples are cleaved by methylation-sensitive restriction endonucleases, such as HpaII and Hin6I, ligated to the CpG-overhang specific adapter, and then cut by McrBC to eliminate residual methylated DNA fragments. The resulting unmethylated DNA fragments are then selectively enriched by adapter-specific aminoallyl-PCR, labelled, and hybridized to microarrays.

 

2. Materials

 

2.1 Adapter design

  1. Adapter storage buffer ST: Prepare 100 ml of 10 mM Tris, pH 8.5; 50 mM NaCl.
  2. Primer for preparation of universal-adapter CG-1: primer CG-1a: 5’-CGTGGAGACTGACTACCAGAT-3’ and primer CG-1b: 5’-AGTTACATCTGGTAGTCAGTCTCCA-3’.

 

2.2 Methylation-sensitive cleavage of DNA

 

1.        Restriction enzymes: 10 U/µl HpaII (Fermentas, Lithuania) and 10 U/µl Hin6I (Fermentas). (Note: HhaI is an isoschizomer of Hin6I, however, the HhaI restriction enzyme is not suitable for adapter-based methylation analyses, since this enzyme does not produce a CpG-overhang that can be ligated to the adapter).

2.   Spike DNA (optional; see Methods)

 

2.3 Ligation

  1. Prepare a 10 µM ATP stock-solution. Make aliquots and store at -20°C.
  2. T4 DNA Ligase (Fermentas, Lithuania)

 

2.4 McrBC digestion

  1. McrBC enzyme from New England BioLabs (NEB).
  2. Guanosine triphosphate (GTP; is delivered with the enzyme): make 10x aliquots, since GTP is highly unstable.

 

2.5 CpG-specific adapter amplification

  1. Labelling nucleotides [20 mM]: Thaw one vial aminoallyl (aa)-dUTP from Ambion, Austin, TX (50 µl solution 5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate). Add 16.5 µl H2O and from 100 mM nucleotide stock solutions (Fermentas) add 16.6 µl dTTP, 41.6 µl dGTP, 41.6 µl dATP, and 41.6 µl dCTP. Mix and store at -20°C.
  2. Taq polymerase [5 U/µl] (NEB).
  3. 200 pmol/µl CG-1b-primer (see 2.1).

 

2.6 Purification of amplification product

  1. Microcon YM-50 columns from Millipore.

 

2.7 Labelling

  1. Sodium bicarbonate buffer (0.1 M, pH 9.0): Prepare 10 ml of a 0.1 M Na2CO3 solution. Add 0.42 g of NaHCO3 to another flask. Add 50 ml of water, and mix with a magnetic stirrer until the NaHCO3 dissolves. The pH should be around 8.5. Adjust the pH to 9.0 by adding ~ 2.5-3.0 ml of the 0.1 M Na2CO3 solution. Monitor the pH change carefully and do not exceed pH 9.3. Filter-sterilize the buffer, which should be prepared as fresh as possible. Aliquot and store at –20°C. Do not re-freeze the buffer. Use every aliquot only once.
  2. Prior to coupling, prepare the dyes (FluoroLink monofunctional dye, Cy3 and Cy5, Amersham Biosciences). The Cy-dyes come in packages, which contain 5 vials. Dissolve 1 vial of dye in 72 µl DMSO. Aliquot 4.8 µl in 15 light protected amber reaction tubes, dry immediately in a speedvac and store at –20°C protected from light. (H2O is also used to dissolve Cy-dyes, however the mono reactive ester is labile in water. Do not use Dioxane, which was suggested elsewhere as an alternative to DMSO. It appears that Cy5 is not fully soluble in Dioxane. DMSO on the other hand is hygroscopic and will absorb moisture from the air, which will react with the NHS ester of the dye and significantly reduces the coupling reaction efficiency. Therefore, keep the DMSO supplied in an amber screw-capped vial at -20°C, and let the vial warm to room temperature before opening to prevent condensation. While the dyes are usually good when first opened, they are sensitive to moisture with a short half-life in aqueous environments. Thus take care to store aliquots of them desiccated, if possible under vacuum).
  3. Quenching buffer: Prepare a 10 ml solution of 4 M hydroxylamine (Sigma). Make aliquots and store at -20°C. Hydroxylamine has top be handled with care, since it may explode when heated. It is an irritant to the respiratory tract, skin, eyes, and other mucous membranes. It may be absorbed through the skin, is harmful if swallowed, and is a possible mutagen.
  4. Purification of dyes: MiniElute PCR-purification kit, Qiagen, Hilden/GER.
  5. Prepare a 3 M NaOAc solution (pH 5.2).

 

2.9 Hybridization

  1. Prehybridization buffer: To 2 ml SlideHyb Glass Array Hybridization Buffer #2 from Ambion add 80 µl of 20 µg/µl yeast tRNA (Sigma) and 200 µg bovine serum albumin (BSA).
  2. Hybridization buffer: To 2 ml SlideHyb Buffer #2 from Ambion (other hybridization buffers may be used. The optimal buffer depends on several experimental variables such as type of glass slide used, the length and GC content of the attached nucleic acid targets, and the manufacturing protocol employed.) Add 50 µl of 20 µg/µl yeast tRNA (Sigma) and 200 µl Cot-1 DNA [1 µg/µl] from Roche Diagnostics. The COT fraction of human placental gDNA consists mainly of repetitive DNA elements. COT DNA is expensive and hence may be omitted if the microarray used does not contain larger amounts of repetitive sequences. If using cDNA arrays, 10-20 µg poly(dA)-poly(dT), which blocks hybridization to polyA tails of cDNA, may be added.
  3. Wash solution I (low stringency): prepare 1 litre of 2x SSC, 0.5 % SDS. Filter-sterilize the buffer. (Note: Wash buffer impurities can cause grainy background; SDS will fluoresce in the Cy5 channel while salt will fluoresce in the Cy3 channel. To eliminate this problem use high quality SDS and SSC for preparation of wash buffers and filter sterilize. Insufficient washing may also contribute to grainy background, therefore, increasing the wash times and/or slight agitation may help to decrease the background).
  4. Wash solution II (high stringency): prepare 1 litre 0.5x SSC, 0.5 % SDS. Filter-sterilize the buffer.
  5. Wash solution III: 1 litre Isopropanol (2-propanol). Use only high quality isopropanol (>99.8%). When it is less concentrated or exposed for a prolonged time to air (it absorbs water quickly), isopropanol causes streaks or residue on the array surface.
  6. Cover slips: Hybri-Slips (Sigma). Hybri-slips have hydrophobic surfaces, which guarantee a constant probe concentration and, unlike glass cover slips, do not adsorb the probes.

 

 

3. Methods

 

Here we present a complete protocol of DNA methylation profiling using the unmethylated fraction of the genome. This protocol works with most microarray types, as exemplified here with CpG island- and oligonucleotide microarrays. A detailed description of how specific ‘epigenetic’ microarrays are designed and how the slides are processed can be found in Schumacher et al., 2006 (1). DNA samples can either be interrogated as pairs (tester and control) on one array, as described in this protocol, or hybridized independently with only one fluorescent dye (e.g. for Affymetrix tiling arrays).

 

3.1 Adapter design

 

In the CpG-specific adapter design, the following aspects must be taken into account:

 

  1. It must contain a CpG-overhang, which fits to the restriction site of the enzymes used (Fig. 2a).
  2. The two nucleotides next to the CpG-overhang have to be different from the nucleotides within the recognition sequence of all the enzymes used in the restriction-digests. This ensures that, after ligation of the adapters, the old restriction-site is disrupted and the sequence cannot be re-cut by the enzymes.
  3. The melting-temperature (Tm) should be about equal throughout the adapter and decreasing at the 3’-end opposite to the CpG-overhang.
  4. The core-sequence of the adapter should be non-homologous to any sequence in the gDNA. Additionally, it is advisable to avoid palindromic sequences, which can lead to mispriming.
  5. If you want to cut the adapter from the target-fragments, include a very specific, new recognition site within the adapters core-sequence (close to CpG-overhang), which does not cut frequently in the genome you analyze. Use a rare 8-mer (e.g. SdaI (5’-CC↑TGCA↓GG) for sticky ends or MssI (GTTT↓AAAC) for blunt ends.
  6. Use a specific, long antisense-overhang, which prevents the forming of tandem-repeats and random blunt-end ligation to the genome (e.g. through degraded DNA).
  7. To avoid improper annealing between the two primers, choose a non-5’-complementary end (Fig. 2a).

 

 

Fig. 2: Preparation of the CpG-specific adapter. a: CG1-adapter components. b: Annealing of the CG-primers produces a double-stranded adapter that can be identified by electrophoresis as a strong band (200 pmol loaded; 2.5 % agarose gel).

 

3.1.1 Adapter preparation

1.        Dissolve the non-phosphorylated ssDNA oligonucleotides in ST buffer with a concentration of approximately 800 pmol. The presence of some salt is necessary for the oligos to hybridize into a double stranded (ds)-adapter. Keep some of the CG-1b oligo, as it will be later used as a primer to amplify the unmethylated DNA fraction. Measurethe concentration in a spectrophotometer and adjust the concentration to 400 pmol. (Note: Quantify in spectrophotometer. Re member the OD260 = 1 of single stranded DNA is equivalent to 33 µg/ml not 50 µg/ml! This relationship, however, can be inaccurate for short fragments of DNA, such as oligonucleotides. Base composition and even linear sequence will affect optical absorbance, hence the precise value of the OD to mass relationship is unique for each oligo. For example, 1.0 OD260 of CCCCCCCCCCCC (homopolymeric deoxycytidine) equals 39 micrograms while 1.0 OD260 of AAAAAAAAAAAA (homopolymeric deoxyadenosine) equals only 20 micrograms. Do not believe the amount of primer as indicated by the manufacqturer. Perform the appropriate calculation (800 pmol primer = 0.0008 x MW of oligo)).

  1. Mix together equal molar amounts of each complementary oligonucleotide to a final concentration of 200 pmol, and heat 5 min in a thermalcycler at 80°C. Cool down with 1°C/min until the mixture reaches room temperature (RT). The longer the cooling period, the lower is the risk of hairpin structures.
  2. Check a small amount of the ds-Adapter on a 2.5% Agarose-gel (see Fig. 2b), which should generate a strong band. Run the single stranded primers next to the adapter as a control.
  3. Store the adapters at –20°C.

 

3.2 Methylation-sensitive cleavage of DNA

 

Over 260 different methylation-sensitive restriction enzymes (MSRE’s; incl. isoschizomers) are now available, however, not all enzymes are useful and informative for DNA methylation profiling. Informative MSREs are defined by the number of cleavage fragments that can be ligated to adapters and efficiently amplified, and are not lost during column-purification steps (3). Some enzymes, although they cut frequently in the genome, produce fewer informative fragments compared to enzymes which do not cut as frequently. For example, the non-palindromic AciI (5’-CCGC-3’) recognizes more than twice as many CpG sites in CpG island regions when compared to HpaII, but on the other hand, produces fewer fragments in the size-range that can be detected by PCR or amplified fragment length polymorphism (AFLP) methods (Table 1).

 

MSRE

Cut site 5’-3’

~% of CpGs in CpG islands*

~% of CpGs in non-CpG islands*

Fragments/kb in CpG islands*

Fragments/kb in non-CpG islands*

AciI (SsiI)

CCGC, GCGG

30.60

17.36

3.23

1.79

Hin6IΔ (HinP1I)

GCGC

14.40

5.05

3.98

0.61

HpaII (BsiSI)

CCGG

11.70

9.33

3.98

1.18

Hin1I (BsaHI)

GRCGYC

2.6

0.9

1.92

0.11

HpyCH4IV (TaiI)

ACGT

1.66

6.73

1.24

0.97

Bsp119I (AsuII)

TTCGAA

0.11

0.13

0.11

<0.02

Bsu15I (ClaII)

ATCGAT

<0.05

0.39

<0.02

0.02

 

Table 1: Methylation-sensitive restriction enzymes. All the above MSRE’s produce sticky ends that can be ligated to the CG-1 adapter for high throughput microarray-based DNA methylation profiling. Other MSRE’s that fit to the CG-1 adapter are Psp1406I, XmiI, BstBI or NarI, however these enzymes have very few restriction sites in the human genome or are too expensive to be used in the required amount. Asterisk (*) indicates the number of 75 bp - 2 kb long, i.e. ‘informative’, fragments, derived from CpG island and non-CpG island sequences in the human genome (3). R = A/G; Y = C/T.

 

Different requirements for the enzyme reduces the list of potentially useful and informative MSRE’s to about 17, which would cover up to 85% of all CpG island CpG dinucleotides, but less than 50% of all CpG dinucleotides in other genomic regions (3). Here, we demonstrate the technology with a double-digestion using HpaII and Hin6I enzymes (Note: To gain the most out of restriction analyses, it is crucial to choose the right enzyme combination for the targets to be interrogated. For example, some MSRE’s cut relative frequently in CpG islands, but rarely recognize a sequence outside of a CpG island region, as is the case for Hin6I (5’-GCGC-3’) or Bsp143II (5’-PuGCGCPy-3’). In contrast, enzymes such as HpyCH4IV (5’-ACGT-3’) cut predominantly outside of CpG island sequences and are less useful in the interrogation of CpG islands, for instance in CpG island-microarray based studies (3). Several other methods rely on the specific methylation-sensitive cleavage of the rare cutter NotI (5’-GCGGCCGC-3’), for example RLGS and AFLP methods and a couple of microarray approaches (6, 7). However, NotI-sites are not well represented in the genome and will only provide a very rough overview of methylation patterns. Hence, it is not advisable to include NotI in genome-wide analyses of e.g. complex diseases).

Before the test DNA can be processed, it is advantageous to add “spiking” DNA to the gDNA samples. The spiking DNA consists of ‘alien’ (exogenous) DNA, which allows monitoring of each step of the experiment (Note: Additionally, spike DNA can be used to test the methylation-sensitive cleavage reaction, ligation efficiency, McrBC digestion and PCR reaction. Spiking DNA is added to the reactions with concentrations equivalent to the template concentration (1 genome equivalent, GE, assuming 3x109 bp in the human genome). For example, 16.2 pg of λ plasmid is added to 1 µg of template DNA. Make 12x GE stock solutions in 1 mM EDTA; aliquot into small volumes and store at -20°C. To prepare a McrBC digestion control, digest pUC57 with HpaII, ligate the CG-adapter and premethylate with SssI methylase. McrBC controls are added to the McrBC cleavage step as premethylated plasmid. Other suitable controls are ФX174 to test for PCR bias or pBR322 to test the ligation efficiency (1). ФX174 DNA has to be digested separately with HpaII/Hin6I and then ligated to the CG-adapter. Complementary spiking oligonucleotides have to be representative sequences of HpaII and Hin6I cleavage fragments).

 

As with any microarray experiment, controls are crucial for monitoring experimental variability. Suitable spiking DNA consists of sequences that are frequently spotted on commercially available microarrays, such as Arabidopsis, RNA spikes or artificial sequences. (Note: SNPs within the recognition sites for HpaII and Hin6I may simulate epigenetic differences (8). In order to exclude the impact of DNA sequence variation it is suggested to check the available SNP databases and identify the DNA sequence variation within the restriction sites of the used enzymes. From CpG island microarray studies, the estimate is that 10 to 30% of methylation variation detected in between individuals could, in fact, be due to DNA sequence variation (1). For comparison, in a study for the Human Epigenome Project (HEP), interrogation of 3,273 unique CpG sites on chromosome 6 revealed that 101 CpGs overlapped with known SNPs (3 %) (9). Another way to differentiate DNA sequence effects from genuine epigenetic differences consists of performing an identical microarray experiment on the same DNA sample that has been stripped of all methylated cytosines (1). To eliminate all methylation, the whole genome has to be amplified (thereby replacing all metC with C), for example using the Phi29 DNA polymerase. Amplified DNA samples are then subjected to the same steps as depicted in Figure 1 and co-hybridized on the microarrays. In this experiment, all of the outliers must be a result of DNA sequence variations within the restriction sites of the enzymes used and can be eliminated from further epigenetic analyses).

 

  1. 400 ng of genomic DNA is digested with 10 U HpaII plus 10 U Hin6I for 6 hours at 37°C. The DNA should be highly concentrated to keep the total volume of the reaction below 20 µl.
  2. The samples are then incubated for 10 min at 65°C to inactivate the enzymes. Let the sample cool down with 1°C/min. This step ensures reannealing of small DNA fragments that can denature during heat inactivation.
  3. Immediately proceed with the ligation reaction. It is important that the digested DNA is processed as soon as possible to avoid degradation of the CpG overhangs.

 

3.3 Ligation

 

  1. Take 200 ng of the digested DNA for a standard ligation. If digested DNA is stored too long before ligation, ligation efficiency will decrease.
  2. Adjust the buffer concentration to 1x, taking into account that the unpurified restriction product still contains salts from the previous DNA cleavage reaction. That means that buffer has to be supplemented for only the extra volume added to the DNA.
  3. Add ATP to a final concentration of 1 mM. Add 120 pmol of the double-stranded CG-adapter and mix. (Note: The amount of CG adapter is directly dependent on the amount of restriction enzymes used. For each enzyme add ~0.3 pmol of the double-stranded CG-adapter per ng DNA. For instance, in a triple digest (HpaII/Hin6I/AciI) of 500 ng DNA it is advised to add 500 x 0.3 pmol x 3 = 450 pmol of the adapter). Adjust the volume of the reaction with water to 18 µl, mix and then heat the sample for 10 min at 45°C in a water bath or thermal cycler (Note: All ligation components have initially combined without the T4-Ligase. Since the CpG-overhang of the adapter is complementary to itself, it could form adapter-dimers during the ligation. Therefore, a heating step is introduced before the ligase is added to the mixture. In this way, adapters will be dissociated to bind specifically to the DNA overhangs produced by the restriction enzymes).
  4. Take the sample out of the cycler and chill on ice immediately. Add quickly 2 µl (10 U) of the ligase to a final volume of 20 µl, mix well without introducing bubbles and place the tubes back into the cycler. Incubate for 16 hours, followed by a short 5 min inactivation step at 65°C. To favour re-annealing of short denatured DNA fragments, let the sample cool down with 1°C/min until room temperature is reached (Note: It is important to reduce the amount of ATP in the mixture, since ATP interferes with the following McrBC digestion by competing with GTP for a binding site in McrBC. Since ATP cannot be utilized by the enzyme to perform the enzymatic reaction, McrBC will bind to the DNA without cleavage. The best way to decrease the amount of ATP is heat degradation, since free ATP is highly unstable).
  5. Store the ligation mixture at –20°C (4°C if the reaction is processed at the same day).

 

3.4 McrBC digestion

 

  1. Perform the reaction with NEB Buffer 2 + 1x BSA [10x] + 2x GTP [2 mM].
  2. Add buffer only for the extra volume of the reaction. The ligation took already place in a restriction enzyme buffer.
  3. ATP inhibits the reaction, therefore make sure that the DNA sample was either purified before McrBC cleavage, or the present ATP was degraded by heating the sample for some time during the “cool down” phase of the ligation procedure. Digest the DNA with 10 U/µg of McrBC for 8 hours at 37°C (Note: McrBC makes one cut between each pair of half-sites, cutting close to one half-site or the other, but cleavage positions are distributed over several base pairs approximately 30 base pairs from the methylated base. Therefore, the enzyme does not produce defined DNA ends upon cleavage. Also, when multiple McrBC half-sites are present in DNA (as is the case with cytosine-methylated genomic DNA) the flexible nature of the recognition sequence results in an overlap of sites, and so a smeared rather than a sharp banding pattern is produced. GTP is more labile than most other nucleotides, so it is recommended to aliquot the 100 mM solution supplied. McrBC will cut the DNA (both strands) even if the methylated cytosines are on only one strand (hemi-methylated).
  4. Heat-inactivate the enzyme for 20 min at 65°C. Store the ligation mixture at –20°C (4°C if the reaction is processed at the same day).

 

3.5 CpG-specific adapter amplification

 

The protocol is based on aminoallyl (aa) nucleotide incorporation followed by coupling to N-hydroxysuccinimide (NHS) functionalized dyes. This indirect labelling method is advantageous compared to direct incorporation of dye labelled nucleotides in gDNA. The aminoallyl-nucleotide is better tolerated by the Taq polymerase compared to other fluorescent nucleotides analogs (Note: This protocol works only with standard Taq polymerases. Other, high-processing polymerases usually can’t incorporate modified nucleotides efficiently enough to produce PCR fragments of the desired length.). While direct incorporation of dye labelled nucleotide protocols typically have incorporation efficiencies in the range of 2-5 dye molecules per 1,000 nucleotides, the aminoallyl protocol can achieve frequencies of incorporation in the range of 10-20 dye molecules per 1,000 nucleotides. Furthermore, the reagent costs for the aminoallyl-NHS protocol are cheaper. Amino allyl-dUTP contains a reactive amino group on a 2-carbon spacer attached to the methyl group on the base portion of dUTP. During the coupling reaction, after DNA synthesis, this amino group reacts with the NHS ester of the monoreactive Cy3 and Cy5 dyes.

 

 

  1. For each 200 ng DNA, prepare the PCR-Mixture as follows (final volume 100 µl):

 

1st BS-PCR

Component

µl

(Amount) & [Conc.]

Comment

H2O

x

 

 

Buffer

10

Contains 15 mM MgCl2

 

MgCl2

14

[25 mM] à3.5 mM MgCl2

 

Allyl-dNTPs

0.5

250 µM

[20 mM]

Primer 1

1

~1pmol for each ng DNA

[200 pmol] Primer CG1b

Primer 2

0

Not necessary

 

Polymerase

3

[5U/µl]

15 U (depending on amount of starting DNA)

Template

x

200 ng

Adapter-ligation product

Enhancer

0

 

 

Total

20

 

 

 

 

 

 

 

2.       Depending on the Thermalcycler, the PCR reaction may have to be split into several tubes. Run the following program:

 

 

Type

Step #

Time

Temp

Cycles

Notes

1st PCR

þ

1

5:00 min

72°C

1x

Fills in 3’ recessed ends

2nd PCR

 

2

1:00 min

95°C

1x

Starting Denaturation

BS-PCR

 

3

0:30 min

93°C

Q

24x

Denaturing

TD-PCR

 

4

2:00 min +

3 sec/cycle

68°C

Annealing & Extension

Notes:

5

5 min

72°C

1x

Final Extension

6

hold

4°C

-

Storage

 

 

  1. Check the size of the amplified DNA by separating 8 µl product on a 1% agarose gel (the size of DNA should range from approximately 150 to 2000 bp; see Fig. 3).

 

 

 

 

 

 

Fig. 3: The adapter-amplification results in a typical smear in the size-range of 150 – 2,000 bp. To some extent, the lengths of the amplified fragments depend on the primer annealing temperature of the PCR reaction. Usually, an increased annealing/elongation temperature will produce larger DNA fragments.

 

3.6 Purification of the amplification product

 

The overall yield of the amplicon-PCR is relatively high (20-100 µg). However, most of the commercially available column-based PCR cleanup kits cannot handle this large amount of DNA without clogging. Therefore, our laboratory uses YM-50 Microcon columns (Millipore) for cleanup, but other systems may work as well (Note: Centrifugation of Microcon YM-50 columns forces liquid through the low-binding cellulose membrane. Solutes larger than the nominal cut-off of the units are retained: YM-50 columns have a double-stranded nucleotide cut-off of 100 bp. This includes about 46 bp from the adapters on both sides of the amplification products. Therefore, all methylation-sensitive restriction products below ~ 54 bp and unincorporated adapters and primers should be removed during cleanup. Recoveries of amplification products are generally above 90 percent. Millipore columns are also available as plates, which could be suitable for high-throughput analysis. Recently, we also used Qiagen columns for purification; the overall amount that can be loaded is lower, however the cleanup will still produce enough material for several hybridizations with high quality DNA.).

 

  1. Add 500 µl nuclease-free water to the empty columns and spin ~6 minutes at 10,000 rpm until the column is dry. (The columns contain small amounts of Tris on their membrane that can inhibit the subsequent labelling reaction. Therefore, the columns have to be washed prior to adding the allyl-PCR product).
  2. Insert the Microcon sample reservoir into a 1.5 ml vial. Pipette the whole PCR-product into the reservoir. Spin per guidelines (approx. 1 min at 10,000 rpm). The exact spin-time is dependent on temperature and rotor and may have to be elucidated empirically. Be sure to align the strap of the collection tube cap towards the centre of the rotor. Following the spin, the membrane should still be slightly wet.
  3. Add 500 µl dd-H2O and spin again 6 minutes at 10,000 rpm. The membrane should be looking wet, but should not contain a visible amount of liquid. If there is liquid, spin it down for another minute.
  4. Add additional 90 µl nuclease free dd-H2O (Note: The following aminoallyl/N-hydroxysuccinimide reaction requires that no free amines be present in the coupling reaction buffer. This means that no Tris buffer can be used during any steps, including the DNA-preparation.).
  5. Place the vial with the column into the Eppendorf shaker and shake at 400 rpm for 1 minute (this helps to elute the entire DNA which is maybe bound to the membrane). If you don’t have an Eppendorf shaker, vortex the column slightly for several seconds.
  6. Uncap the Microcon unit. Separate the sample reservoir from the filtrate cup, and place the sample reservoir upside down into a new vial. Spin for 3 minutes at 4000 rpm in invert spin mode to elute DNA.
  7. Remove the column. Cap the vial with the eluate (~100 µl) for storage at –20°C.
  8. Use 5 µl of the eluate to measure the DNA concentration in a spectrophotometer.

 

3.7 Labelling

 

3.7.1 Coupling

The amount of unmethylated DNA fraction to be hybridized to an array depends on the surface area of the microarray. In our experience, 2 µg of the eluate is enough for a standard slide.

 

  1. For all steps: Shield all samples, which contain fluorescent-dyes from light!
  2. Dry your DNA sample (2 µg) in a speedvac.
  3. Resuspend the DNA pellet in 9 µl of the 0.1 M Sodium-Bicarbonate Buffer. Add 3 µl DMSO. Let sample sit for 10-15 minutes.
  4. Denature the sample for 2 min at 100°C. In our experience, single stranded DNA incorporates the Cy-dyes better compared to native DNA.
  5. Quickly spin down and add the sample to a dry aliquot of monofunctional Cy3 or Cy5 dye. Resuspend the dye pellets by pipetting carefully; try not to introduce air bubbles (if it happens, centrifuge the sample for a few seconds, this will get rid of the bubbles).
  6. Incubate for 2 hours at 30°C in the dark.

 

3.7.2 Quenching

After the coupling step, reactive groups on the dyes must be quenched to prevent cross reactivity or exchange of dye molecules between active aminoallyl nucleotides. This protocol uses hydroxylamine to quench.

 

  1. Add 4.5 µl of 4 M hydroxylamine. Mix well by pipetting up and down.
  2. Incubate for 15 minutes at RT (Eppendorf shaker, 400 rpm) in the dark.
  3. Combine the Cy3 and C5 samples.

 

3.7.3 Cleanup of product

To remove unincorporated dyes, columns have to be applied that efficiently purify fragments in the size range of ~ 50 – 2,000 bp. For this protocol, we are using the Qiagen MiniElute PCR-purification kit.

 

  1. Add 25 µl of dd-H2O to the sample.
  2. Add 3 µl of 3 M NaOAc (pH 5.2) to ensure a low pH of the mixture. It is important to keep in mind the DNA binding curve for silica, on which this kit is based, is favourable at low pH but falls off precipitously around pH 8. Thus it is essential that the pH of your reaction be below pH 7.5.
  3. Add 275 µl (5x Vol.) of Buffer PB and mix. Apply to column and centrifuge at 13,000 rpm for 1 min.
  4. Discard the flow-through and wash the DNA five times with 750 µl Buffer PE at 13,000 rpm for 1 min. A critical step for minimizing background is to effectively wash away all unbound dye molecules during the final cleanup; small amounts of unincorporated dye can have a large absorbance and thus give misleading results. If the colour from the dyes is still visible in the wash, continue running wash buffer through the column until the eluate is clear.
  5. Centrifuge the column for an additional 1 min at maximum speed.
  6. Place the column in a new, clean 1.5 ml microcentrifuge tube and elute by adding 25 µl prewarmed (~50°C) elution buffer (EB) to the centre of the membrane. Let it stand for 1 min at RT and spin at 13,000 rpm for 1 min.
  7. Repeat the elution step with an additional 25 µl (final volume 50 µl).

 

3.7.4 Determination of incorporated dyes

  1. Place the entire 50 µl sample in a clean microcuvette. Measure the absorbance of the Cy3/Cy5 sample in a spectrophotometer at 260 nm (DNA), 550 nm (Cy3 fluorescence) and 650 nm (Cy5 fluorescence). Be very careful about contamination in the cuvette as you will be recovering the sample for hybridization. Wash carefully after each measurement.
  2. Calculate the amount of recovered DNA: A260 x 50 x total volume of sample (µl) = ng of target.
  3. Calculate the frequency of incorporation (FOI): For Cy3 incorporation 86.5 x (A550/A260) and for Cy5 incorporation 51.9 x (A650/A260). The results are expressed as the number of Cy-dCTP incorporated per 1.000 nucleotides of DNA (Note: The numbers 86.5 and 51.9 are conversion factors calculated by using the average molecular weight of dNTPs (324.5 g/mole), the absorption coefficient of Cy3 and Cy5 (150,000/M*cm and 250,000/M*cm) and the concentration of Cy-labelled allyl-PCR products that absorbs 1 AU of 260-nm light (50 µg/mL). When measuring ss-DNA, the concentration of Cy-labelled ss-DNA that absorbs 1 AU of 260-nm light is 37 µg/mL. The conversion factors changes then to 117 for Cy3 and 70.2 for Cy5. Note that sample volume is not factored in; this is because volumes cancel out when DNA and cyanine absorbance are measured simultaneously. Frequently, the FOI for the different dyes may differ. In our experience, Cy5 is not as well incorporated as Cy3, however, it really depends on several factors (humidity, ozone concentration, etc) and that is why it is recommended to do reciprocal labelling). Optimal frequencies of incorporation are between 15-20 and higher, though anything higher than 10 will give satisfactory results. Using targets with less than FOI 6 may give high background or very weak signals.

 

3.8 Hybridization

 

Before starting to hybridize, it is recommended that the cover slips are already cut, the hybridization chambers ready, and that all solution are prepared and adjusted to the correct temperature. Also, scan one array prior to hybridization to determine basal level of background on the array (Note: As arrays age, some surface chemistries, particularly poly-L-lysine, show an increase in auto-fluorescence in the Cy3 channel. Aged microarrays may also show a higher affinity for salt, which will fluoresce in the Cy3 channel. To minimize these effects use the arrays as fresh as possible and store them properly (in the dark and under vacuum, if possible).

 

3.8.1 Preybridization

  1. Briefly heat the Prehyb solution in an amber reaction tube to 72°C and let it cool down to RT.
  2. To locate the area on the microarray that has to be hybridized, place the dry microarray on a hybridisation template that shows the dimensions of the slide and the area where the grids are printed.
  3. Add water into the hybridization chamber to prevent drying of the samples.
  4. Pipette the Prehyb-solution to the designated area of the slide. Avoid surface contact with objects such as pipette tips. If any bubbles appear, try to remove these with a syringe needle.
  5. When bubble-free, apply the cover slip carefully (use cover slips larger then the grid-area). Use tweezers.
  6. Put the slide into a hybridization chamber and pre-hybridize the array for 1 hour at 45°C.
  7. After incubation, move the slides to a 45°C jar containing dd-water and remove the cover slip carefully, without using force.
  8. Transfer the array into a new jar containing 45°C dd-water and leave them there for 2 minutes, moving the slide up and down several times. Repeat wash step 2 times.
  9. Transfer the slide again into a new jar containing RT isopropanol and repeat the washing by moving the slide up and down several times (~1 min).
  10. Immediately blow-dry the slide with pressurized air. The pre-hybridized arrays can now be hybridized or stored in a dark place for later usage.

 

3.8.2 Hybridization

  1. Place the complete 50 µl DNA sample in a speedvac (shield from light) until the solution is reduced to about 5 µl. Do not allow the labelled DNA to dry completely!
  2. Preheat the Hyb-solution prior to use for 5 minutes at 72°C. Make sure the Hyb-solution is thoroughly resuspended. Centrifuge for 1 minute at 13,000 rpm to get rid of particulate material. Pipette the needed amount (~60 µl for a 12,200 feature CpG island array) of the Hyb-solution and keep it at RT for 10 min.
  3. Add your DNA (~5 µl) to the Hyb-Mix, denature it for 5 minutes at 80°C and briefly spin down.
  4. Add water into the hybridization chamber to prevent drying of the samples.
  5. Pipette the Hyb-solution to the designated area of the slide.
  6. Apply the cover slip carefully to the bubble-free Hyb-solution. Again, avoid any contact with the array-surface.
  7. Place the slide quickly, but carefully, into a hybridization chamber to prevent renaturation of the probe and evaporation of the Hyb-solution. Make certain that the array surface is level.
  8. Protect the Hyb-chambers from light, and hybridize for approx. 12-16 h at 50°C. (Oligo arrays may require a lower temperature, whereas higher temperatures are needed for BAC/PAC arrays).

 

3.8.3 Washing

  1. Fill three jars with Wash I and two jars with Wash II and heat all to 50°C.
  2. Remove the cover slip by rinsing the slide carefully in Wash I (Jar 2). Take extreme care when handling the array. Allow cover slip to float off the surface. Do not attempt to force off the cover slip as this will likely damage the array.
  3. Place the array slide quickly into the second Wash I (Jar 2) for 15 minutes (all wash steps in the dark), moving the slide up and down from time to time.
  4. Transfer the slides to a new jar (Jar 3) and repeat the wash step for 15 min.
  5. Place the array slide into Wash II (Jar 4) for 15 minutes, moving the slide up and down from time to time.
  6. Transfer the slides to a new Wash II (Jar 5) and repeat the wash step (15 min).
  7. Rinse the slide for 2 minutes within another jar in dd-water (Jar 6) to remove all traces of SDS and SSC. Transfer the slide into a new jar with isopropanol (Jar 7) for 30 seconds, moving it up and down (Note: Salt from the final wash buffer may precipitate out of solution onto the array surface upon submersion into isopropanol. This is likely a result of a higher than intended concentration of salt in the wash-buffer; accidentally switching the wash buffers can cause this phenomenon. A brief re-wash in water followed by another isopropanol wash will likely remove the salt from the array).
  8. Blow the slide dry as quickly as possible using pressurized air. Any buffer left on the array will appear as haze on array and result in a strong green background.
  9. Scan the array in the Cy3 and Cy5 channel (532 nm and 635 nm).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

References

 

1.         Schumacher, A., Kapranov, P., Kaminsky, Z., Flanagan, J., Assadzadeh, A., Yau, P., Virtanen, C., Winegarden, N., Cheng, J., Gingeras, T., and Petronis, A. (2006) Microarray-based DNA methylation profiling: technology and applications. Nucleic Acids Res 34, 528-542.

2.         Cheng, J., Kapranov, P., Drenkow, J., Dike, S., Brubaker, S., Patel, S., Long, J., Stern, D., Tammana, H., Helt, G., Sementchenko, V., Piccolboni, A., Bekiranov, S., Bailey, D. K., Ganesh, M., Ghosh, S., Bell, I., Gerhard, D. S., and Gingeras, T. R. (2005) Transcriptional maps of 10 human chromosomes at 5-nucleotide resolution. Science 308, 1149-1154.

3.         Schumacher, A., and Petronis, A. (2006) Epigenetics of complex disease: from general theory to laboratory praxis. Curr Top Microbiol Immunol 310, 81-115.

4.         Schumacher, A. (2001) Mechanisms and brain specific consequences of genomic imprinting in Prader-Willi and Angelman syndromes. Gene Funct. Dis. 1, 7-25.

5.         Schumacher, A., Arnhold, S., Addicks, K., and Doerfler, W. (2003) Staurosporine is a potent activator of neuronal, glial, and "CNS stem cell-like" neurosphere differentiation in murine embryonic stem cells. Mol Cell Neurosci 23, 669-680.

6.         Yamamoto, F., and Yamamoto, M. (2004) A DNA microarray-based methylation-sensitive (MS)-AFLP hybridization method for genetic and epigenetic analyses. Mol Genet Genomics 271, 678-686.

7.         Li, J., Protopopov, A., Wang, F., Senchenko, V., Petushkov, V., Vorontsova, O., Petrenko, L., Zabarovska, V., Muravenko, O., Braga, E., Kisselev, L., Lerman, M. I., Kashuba, V., Klein, G., Ernberg, I., Wahlestedt, C., and Zabarovsky, E. R. (2002) NotI subtraction and NotI-specific microarrays to detect copy number and methylation changes in whole genomes. Proc Natl Acad Sci U S A 99, 10724-10729.

8.         Schumacher, A., Friedrich, P., Schmid, J., Ibach, B., Eisele, T., Laws, S. M., Foerstl, H., Kurz, A., and Riemenschneider, M. (2006) No association of chromatin-modifying protein 2B with sporadic frontotemporal dementia. Neurobiol Aging, Sept 14, epub

9.         Rakyan, V. K., Hildmann, T., Novik, K. L., Lewin, J., Tost, J., Cox, A. V., Andrews, T. D., Howe, K. L., Otto, T., Olek, A., Fischer, J., Gut, I. G., Berlin, K., and Beck, S. (2004) DNA methylation profiling of the human major histocompatibility complex: a pilot study for the human epigenome project. PLoS Biol 2, e405.