DNAmethyltransferaseAssay

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 NH₄Oac (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 1/10^th 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 -20^oC 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 ml dNTP mix (25mM), 0 m l (RXMA) or 5 m l (RMCA) DMSO, H₂O 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 80^oC 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 ³²P 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 methyl