Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2284-9.

Establishment of methylation-sensitive-representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors.

Ushijima T, Morimura K, Hosoya Y, Okonogi H, Tatematsu M, Sugimura T, Nagao M.

Source

Carcinogenesis Division, National Cancer Center Research Institute, Tokyo, Japan.

Abstract

Methylation of CpG sites in the genome, which is generally conserved during cell replication, is considered to play important roles in cell differentiation and carcinogenesis. However, investigations on changes in methylation status have been limited to known genes. To make a genome-wide search for differentially methylated genes, we developed a methylation-sensitive-representational difference analysis (MS-RDA) method. The representation of the genome was prepared using the methylation-sensitive restriction enzyme HpaII, and the mixture ratio of tester and driver DNAs was optimized to detect differences in methylation status of a single copy per diploid mammalian genome. By performing comparative MS-RDA of one hepatocellular carcinoma and of background liver tissue of one mouse treated with a food carcinogen (2-amino-3,4-dimethylimidazo[4,5-f] quinoline), we were able to identify (i) extensive hypomethylation of long interspersed nuclear element repetitive sequences in a number of hepatocellular carcinomas, (ii) reduction of the gene dosage of their mitochondrial DNA, and (iii) a hypermethylated DNA fragment of unknown origin. Furthermore, by adding the clones obtained in the first MS-RDA to the driver DNA [MS-RDA with elimination of excessive clones (MS-RDA-WEEC)], nine DNA fragments that could not be detected at the first MS-RDA were isolated as differentially methylated DNA fragments. MS-RDA, combined with MS-RDA-WEEC, is thus a promising approach to identify DNA fragments differentially methylated in two DNA sources.

Comment in

Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2103-5.

PMID:: 9122186; [PubMed - indexed for MEDLINE]
PMCID: PMC20079

Free PMC Article

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Figure 1

Optimization of MS-RDA conditions. (A and B) Ethidium bromide staining. (C and D) Southern blot hybridization probed with pN3. (A and C) Amplicons (lanes 5 and 6; 4 μg/lane) were prepared from HpaII digests of mouse liver DNA supplemented with unmethylated pN3 or methylated pN3 (lanes 1 and 2; 10 μg). Lanes 3 and 4: MspI digests of the same DNA with added pN3 (10 μg). Samples were run in 1.1% agarose and blotted. Fragments that were derived from the HpaII digest of pN3 and enriched in the amplicon U are indicated by closed arrowheads (C, lane 5). (B and D) By varying the amounts of tester DNA used for the first, second, and third cycles of competitive hybridization, the optimum allowing detection of a difference of methylation state of one copy per diploid mouse genome was established. Lanes 7 and 8: 2 μg of amplicons prepared from liver total DNA with unmethylated and methylated pN3, respectively (the same as with the amplicon in lanes 5 and 6). Lanes: 9–11, 1 μg of C1-1000, C1-200, and C1-40; 12–16, 1 μg of C2-200, C2-100, C2-40, C2-10, and C2-2; 17–25, 1 μg of C3a-20, C3a-7, C3a-2, C3b-20, C3b-7, C3b-2, C3c-20, C3c-7, and C3c-2; 26: 375 ng of DNA size marker φX174/HaeIII digest; and 27, 100 ng of HpaII digest of pN3. Samples were run in 2% NuSieve/GTG (FMC) agarose and blotted. Repetitive sequences enriched during the competitive hybridizations are indicated by closed arrowheads (B, lanes 9–15). Three target fragments enriched in the amplicons (D, lane 7, shown with closed arrowheads) and another ≈300-bp fragment were enriched by two cycles of competitive hybridization. Target fragments recovered as visible bands are shown by open arrowheads (B and D, lanes 12–14 and 17–22). For a detailed description, see Results. Am., amplicon.

Establishment of methylation-sensitive–representational difference analysis and isolation of hypo- and hypermethylated genomic fragments in mouse liver tumors

Proc Natl Acad Sci U S A. Proc Natl Acad Sci U S A;94(6):2284-2289.