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Methylated CpG island Amplification
CpG Island Methylation in Aging and Cancer  

  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







RMCA primers







3. Methods

3.1. Preparation of MCA amplicons

3.1.1 Digestion of genomic DNA

3.1.2 Ligation of adapter

3.1.3 PCR amplification


3.2. Detection of Aberrant Methylation by Dot blot Analysis

3.2.1 Preparation of filters

3.2.2 Hybridization


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

3.3.3 Change of adaptors on the tester amplicon.

3.3.4 Competitive hybridization.

3.3.5 Selective amplification

3.3.6 Second round subtraction.

3.3.7. Identification of differentially methylated sequences.

4. Notes

  1. 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.
  2. 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).
  3. 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.


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