Methylation-sensitive amplicon subtraction: a novel method to isolate differentially methylated DNA sequences in complex genomes
✍ Scribed by Knut Müller; Walter Doerfler
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 288 KB
- Volume
- 1
- Category
- Article
- ISSN
- 1438-7506
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✦ Synopsis
DNA sequences in complex genomes
of Cologne, Koeln, Germany A new protocol, termed methylation-sensitive amplicon subtraction (MS-AS), has been developed for the identification and cloning of aberrantly methylated DNA segments in complex genomes. This method is based on the preparation of amplicons from HpaII fragments and combines the normalization of relative DNA sequence abundancy and subtractive hybridization. The normalization step applies a specific form of PCR that permits the exponential amplification of differences in HpaII fragment representations, whereas amplification of common sequences is repressed. In contrast to other subtractive hybridization protocols for genomic DNA analyses, the MS-AS method requires only one cycle of competitive hybridization for the efficient enrichment of target molecules. MS-AS analyses of adenovirus type 12 (Ad12)-transformed or bacteriophage lambda DNA-transgenic hamster cell lines have led to the identification of several CpG-rich cellular gene fragments the methylation of which has been altered in the transgenic as compared to the nontransgenic cell lines. The new method adds to the growing number of genome scanning approaches, including methylation-sensitive representational difference analysis, restriction landmark genomic scanning, and methylation-sensitive arbitrarily primed PCR which all have been used to detect altered methylation sites in cancer cells.