DNA methyltransferase Assay

Methylated CpG island Amplification

Protocol written by Minoru Toyota

2. Materials

2.1. MCA

Restriction enzymes SmaI, XmaI

T4 DNA ligase

Taq DNA polymerase

10X PCR reaction buffer:

670mM Tris-HCl, pH 8.8

40mM MgCl2

160 mM NH4 (SO4)2

100 mM b -Mercaptoethanol

1 mg/ml bovine serum albumin.

Tris-EDTA (TE) pH 8.0

DNA precipitation reagents:

Phenol/Chloroform pH 8-9

3M NaOAc (for general precipitation)

5M NH4 Oac (for precipitation and quantitation when dNTPs are present)

100% ETOH

Agarose gel electrophoresis reagents

Filter hybridization reagents:

96 pin replicator system (Nunc)

Nylon membranes

DNA hybridization solution (e.g. BLOTTO)

Random-primed DNA labeling kit

Wash solutions (Wash1 2xSSC, 0.1%SDS; Wash2 0.1XSSC, 0.1%SDS)

2.2. RDA and cloning PCR products.

3 X EE buffer : 30 mM EPPS (SIGMA) pH 8.0, 3 mM EDTA pH 8.0.

5 M NaCl

cDNA spun column (Amersham)

Mung bean nuclease (NEB)

pBluescript (Stratagene)

2.3 Oligonucleotides.

RXMA primers

RXMA24 : 5’-AGCACTCTCCAGCCTCTCACCGAC-3’

RXMA12 : 5’-CCGGGTCGGTGA-3’

JXMA24 : 5’-ACCGACGTCGACTATCCATGAACC-3’

JXMA12 : 5’-CCGGGGTTCATG-3’

NXMA24 : 5’-AGGCAACTGTGCTATCCGAGTGAC-3’

NXMA12 : 5’-CCGGGTCACTCG-3’

RMCA primers

RMCA24 : 5’-CCACCGCCATCCGAGCCTTTCTGC-3’

RMCA12 : 5’-CCGGGCAGAAAG-3’

JMCA24 : 5’-GTGAGGGTCGGATCTGGCTGGCTC-3’

JMCA12 : 5’-CCGGGAGCCAGC-3’

NMCA24 : 5’-GTTAGCGGACACAGGGCGGGTCAC-3’

NMCA12 : 5’-CCGGGTGACCCG-3’

3. Methods

3.1. Preparation of MCA amplicons

3.1.1 Digestion of genomic DNA

• • Digest 5 m g of genomic DNA using 100 units of SmaI over night.
  • Add 20 units of XmaI and incubate at 37 ° C for 6 hours.
  • Add one volume PC9, vortex, spin and extract the supernatant.
  • Precipitate the DNA: Add 1/10th volume 3M NaOAc and 2 volumes 100% ETOH. Store at �70 ° C for 1 hour and centrifuge 30 min. at >10,000 g. Pour the ETOH out, air dry the pellets.
  • Resuspend in 10-20 m l TE and determine DNA concentration using a spectro-photometer.

3.1.2 Ligation of adapter

• • Prepare an adaptor mixture by diluting the primers to 100 m M and combining 50 m l of RXma24 with 50 m l RXma12 (or RMCA24 and RMCA12).
  • Incubate at 65 ° C for two minutes and cool to room temperature for 30 to 60 min. This mixture can be stored at -20o C for up to 6 months.
  • Mix the following: 500 ng of Digested DNA, 10 m l of adaptor mixture, 400 Units of T4 DNA ligase, 3 m l of 10X ligase buffer and water to a total volume of 30 m L.
  • Incubate at 16 ° C for 3-16 hours.

3.1.3 PCR amplification

• • Prepare tubes containing 10 m l of 10 X PCR buffer, 100 pmol of RXMA24 (or RMCA24) primers, 15 Units of Taq DNA polymerase, 1.2 m l dNTP mix (25mM), 0 m l (RXMA) or 5 m l (RMCA) DMSO, H2 O to a total volume of 97 m l.
  • Add 3 m l of ligation mixture. Cover with mineral oil.
  • To fill the 3’-recessed ends of the ligated fragments, incubate at 72° C for 5 min.
  • Perform 25 cycles of PCR (95° C for 1 min and 72° C (for RXMA24) or 77° C (for RMCA24) for 3min), with a final extension time of 10 min.
  • After the reaction, electrophorese 10 m l of the PCR products in a 1.5% agarose gel to check the quality of the amplification. You should see a relatively strong smear, ranging from 300 bp to 2 kb.

3.2. Detection of Aberrant Methylation by Dot blot Analysis

3.2.1 Preparation of filters

• • Transfer the PCR products to a new tube and add 2/3 volume of 5M NH4OAc and 350 m l of 100% ethanol.
  • Chill at �70 ° C for 1 hour and precipitate DNA by centrifugation. Resuspend DNA in 10-15 m l TE buffer and quantitate in a spectrophotometer.
  • Dilute India Black Ink by adding 20 m l to 10 ml H2O. Add 20 m l of this diluted solution to 10 ml 20xSSC.
  • Dilute 1 m g of MCA amplicon in TE (total volume 4 m l). Add 2 m l of the 20xSSC/India Ink solution.
  • Blot (in duplicate) onto nylon membranes. The easiest way to do this is to transfer the DNA mix to a 96 well plate and use a 96 pin replicator system (Nunc). Dry the membrane at RT for 30 min.
  • Place the filters in 0.5M NaOH/ 1.5M NaCl solution for 5 min.
  • Place the filters in 0.5M tris-HCl, pH 8.0 / 1.5M NaCl. Neutralize for 5 min.
  • Transfer the filters to 3M SSC and rinse for 5 min.
  • Dry the filters at RT for 1 hour.
  • Cross link the DNA to the filters using a UV cross linker (or bake at 80o C for 30 min.).

3.2.2 Hybridization

• • Prehybridize filters in a DNA prehybridization solution such as Blotto at 65° C for 3 hours.
  • Label 20 ng of the probe using random priming and 32 P dCTP. Boil the probe, cool on ice and add to the hybridization solution.
  • Hybridize filters for 12-16 hours.
  • Wash with 2xSSC, 0.1% SDS at 65 ° C for ten min. twice, and 0.1XSSC, 0.1% SDS at 65° C 20 min.
  • Expose the filters to a phosphor screen (or use conventional film autoradiography).
  • Develop after 1-3 days exposure. See examples of results in Fig. 2.

3.3 MCA coupled with RDA

3.3.1 Outline

For detection of differentially methylated sequences, you need to generate MCA amplicons from the tester samples (e.g. a cancer sample) and relatively large quantities of MCA amplicons from the driver samples (e.g. DNA from normal tissues) with the adaptors removed. The tester’s adaptors will then be changed and the DNA hybridized with driver DNA, followed by PCR amplification using the second set of adaptors. The subtraction is then repeated once, and the resulting amplicons are further cloned and characterized.

3.3.2 Removal of adaptors from the driver amplicon

• • Perform MCA on multiple aliquots of driver DNA. We typically run 10 reactions in parallel. Verify and pool the aliquots. Quantitate in a spectrophotometer.
  • Digest the driver MCA amplicons using 2 Units/m g SmaI to remove the RMCA/RXMA adaptor. Inactivate SmaI by phenol/chloroform extraction.
  • Remove the adaptors using a cDNA Spun column (Amersham).
  • Electrophorese an aliquot of DNA before and after column filtration to check for complete elimination of adaptors.
  • Add 1/30th volume of 3M Sodium acetate, two volumes of ethanol, chill at �70 ° C, precipitate by centrifugation, resuspend in 200-400 m l TE and quantitate by spectrophotometry.
  • 80 m g of processed driver DNA is required per tester sample (per condition).

3.3.3 Change of adaptors on the tester amplicon.

• • Digest 5 m g of tester MCA amplicons using 20 Units XmaI to remove the RMCA/RXMA adaptor. Inactivate XmaI by phenol/chloroform extraction.
  • Remove the adaptors using a cDNA Spun column (Amersham).
  • Electrophorese an aliquot of DNA before and after column filtration to check for complete elimination of adaptors.
  • Add 1/30th volume of 3 M Sodium acetate, two volumes of ethanol, chill at �70 ° C, precipitate by centrifugation, resuspend in 10-20 m l TE and quantitate by spectrophotometry.
  • Prepare JMCA and/or JXMA adaptors as described in section 3.1.2. Ligate the adaptors to 0.5 m g of the above DNA as described in section 3.1.2.

3.3.4 Competitive hybridization.

• • Add 70 m l of TE to the ligation mix and purify DNA by phenol/chloroform extraction. Mix all of this DNA with 40 m g of MCA amplicons from driver DNA.
  • Add 1/10th volume of 3 M NaOAc and 2 volumes of ethanol, chill at �70 ° C for 30 min, and centrifuge for 30 min.
  • Dissolve DNA in 4 m l of 3 X EE (30 mM EPPS, 3 mM EDTA) solution and transfer to a 0.5 ml microcentrifuge tube.
  • Denature DNA at 96 ° C for 10 min, quick spin, add 1 m l of 5 M NaCl, cover with mineral oil and incubate at 67 ° C for 20 hours (this is best done in a thermocycler).

3.3.5 Selective amplification

• • Heat 100 m l of 1 M NaCl at 67 ° C for 5 min., and add 45 m l to the competitive hybridization solution.
  • Prepare PCR mixture as follows : 10 m l of 10X PCR buffer, 5 m l (1/10th ) of the hybridization mix, 1.2 m l of 25 mM dNTP mix, 100 pmol JXMA24 (or JMCA24) primers, 15 Units of Taq DNA polymerase, 0 m l (RXMA) or 5 m l (RMCA) DMSO, H2 O to a total volume of 100 m l. Cover with mineral oil.
  • Fill the ends at 72 ° C for 5 min. PCR amplification is then done at 10 cycles of 95° C for 1 min and 72° C (JXMA24) or 77° C (JMCA24) for 3 min. Final extension is at 72° C for 10 min.
  • Transfer the PCR products to a clean microcentrifuge tube.
  • To digest the single stranded amplified MCA products, add 10 m l of 10X Mung bean nuclease buffer and 100 units of Mung bean nuclease, and incubate at 30° C for 30 min.
  • Purify DNA with phenol/chloroform extraction, add 2/3 volume of NH4 OAc, and two volumes of ethanol, chill at �70 ° C for 30 min. and precipitate by centrifugation.
  • Resuspend DNA in 50 m l water. Add 10 m l of 10X PCR buffer, 1.2 m l of 25 mM dNTP mix, 100 pmol JXMA24 (or JMCA24) primers, 15 Units of Taq DNA polymerase, 0 m l (RXMA) or 5 m l (RMCA) DMSO, H2 O to a total volume of 100 m l. Cover with mineral oil.
  • PCR amplification is then done at 20 cycles of 95° C for 1 min and 72° C (JXMA24) or 77° C (JMCA24) for 3 min. Final extension is at 72° C for 10 min.
  • After the reaction, electrophorese 10 m l of the PCR products in a 1.5% agarose gel to check the quality of the amplification. You should see a DNA smear, ranging from 200 bp to 1 kb.

3.3.6 Second round subtraction.

• • Remove JMCA/JXMA adaptors and ligate NMCA/NXMA adaptors as described in section 3.3.3
  • For the second round of subtraction 100 ng of tester and 40 m g of driver are used. Hybridize and proceed with PCR as outlined in sections 3.3.5 and 3.3.5.
  • Clone the MCA products obtained after two rounds of subtraction and amplification: DNA is digested with XmaI, purified by phenol/chloroform extraction and ethanol precipitation.
  • Resuspend the DNA in water and ligate to pBluescript (or your favorite cloning vector) digested with XmaI and treated with calf intestinal phosphatase. Transform appropriate strains of bacteria, grow overnight and screen the colonies for the presence of inserts.

3.3.7. Identification of differentially methylated sequences.

• • At this point, many of the inserts are Alu repetitive elements, and some are false positives.
  • To identify non-repeated true positives, we first amplify the inserts by PCR and screen them by dot-blot hybridization using an Alu probe.
  • All non-Alu hybridizing inserts are then used as probes on filters containing MCA amplicons from tester and driver.
  • All inserts that detect a strong dot in the tester and a weak (or absent) dot in the driver are sequenced and further characterized by Southern blotting and other techniques.

4. Notes

CpG islands vary in their CpG density such that different PCR primers and conditions may be required for effective MCA amplification. For this reason, we have developed two different sets of primers (RMCA and RXMA) that differ in CG content and represent slightly different subsets of CpG islands. On average, the RMCA primers amplify smaller and more CG rich fragments than RXMA. Some probes work well using either condition, but others work better (or exclusively) using one of the sets of primers. For example, the P16 CpG island amplifies better with RMCA. Thus, to adapt this procedure to a known gene, one has to try both RMCA and RXMA, probe with a DNA fragment that is contained between 2 SmaI sites in the gene of interest, and determine the optimal condition. In some cases, it may be required to change the PCR conditions and/or primers. Similarly, for cloning differentially methylated CpG islands, we have used both RMCA and RXMA amplicons, and have obtained a different spectrum of sequences. It is highly recommended to add positive and negative controls for MCA on every filter. For semi-quantitative detection of methylation by MCA, we also recommend adding a mixture of the positive and negative controls on every filter. For example, we usually use a mixture of a colon cancer cell line (Caco2) and normal colon DNA, where the proportion of the cancer DNA (which is methylated at the locus of interest) is 100%, 50%, 10%, 1%, 0.1% and 0%. The intensity of hybridization for the unknown samples can then be compared to this dilution curve (see Fig. 2). In our experience, 70-90% of the amplified MCA products represent Alu sequences. After two rounds of subtraction, 50-70% of the subtracted fragments also contain Alu sequences. This may be due to the fact that some Alus are differentially methylated in cancer. When excluding Alu sequences, 70-80% of the fragments were true positives i.e. differentially methylated. The rest were sequences methylated in both testers and drivers. Using MCA/RDA, we have not recovered sequences that are unmethylated in both tester and driver.