Analysis of repetitive element DNA methylation by MethyLight
By D. Weisenberger, Mihaela Campan, T. Long, Myungjin Kim, Christian B. Woods, E. Fiala, Melanie Ehrlich, Peter W. Laird. Nucleic Acids Research, 2005
727 citations recorded by the source corpus.
Repetitive elements represent a large portion of the human genome and contain much of the CpG methylation found in normal human postnatal somatic tissues. Loss of DNA methylation in these sequences might account for most of the global hypomethylation that characterizes a large percentage of human cancers that have been studied. There is widespread interest in correlating the genomic 5-methylcytosine content with clinical outcome, dietary history, lifestyle, etc. However, a high-throughput, accurate and easily accessible technique that can be applied even to paraffin-embedded tissue DNA is not yet available. Here, we report the development of quantitative MethyLight assays to determine the levels of methylated and unmethylated repeats, namely, Alu and LINE-1 sequences and the centromeric satellite alpha (Satα) and juxtacentromeric satellite 2 (Sat2) DNA sequences. Methylation levels of Alu, Sat2 and LINE-1 repeats were significantly associated with global DNA methylation, as measured by high performance liquid chromatography, and the combined measurements of Alu and Sat2 methylation were highly correlative with global DNA methylation measurements. These MethyLight assays rely only on real-time PCR and provide surrogate markers for global DNA methylation analysis. We also describe a novel design strategy for the development of methylation-independent MethyLight control reactions based on Alu sequences depleted of CpG dinucleotides by evolutionary deamination on one strand. We show that one such Alu-based reaction provides a greatly improved detection of DNA for normalization in MethyLight applications and is less susceptible to normalization errors caused by cancer-associated aneuploidy and copy number changes.
Subfields: Molecular Biology
View Paper: https://www.semanticscholar.org/paper/b0981bba15814b8bb16875abc67c28c0cc160db0
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In Molecular Biology
- Peter W. Laird
- P. Molloy
- Andrea A. Baccarelli
- B. Power
- Ann Wilson
- Manel Esteller
- Jian‐Kang Zhu
- Christoph Bock
Citations in the corpus
- Rapid DNA methylation changes after exposure to traffic particles. (2009, background): “Never smoker 338 (31) 214 (30) 115 (33) 9 (31) Former smoker 714 (65) 476 (66) 219 (62) 19 (65) Current smoker 45 (4) 28 (4) 16 (5) 1 (4) Cumulative smoking,* pack-years (SD) 30.”
- Principles and challenges of genome-wide DNA methylation analysis (2010, methodology): “However, a more efficient method relies on the recognition that a SNP caused by evolutionary deamination of me C to T will have been propagated on the opposing DNA strand as an A, whereas bisulphite deamination of an unmethylated cytosine will leave the G on the opposing strand unaffecte”
- DNA hypomethylation and human diseases. (2007, methodology): “Single gene hypermethylation studied in same samples HPLC %meC of total C [110] Wilms tumors: No hypomethylation (n=12) 3.57±0.12% Moderate hypomethylation (n=10) 3.32±0.07% Strong hypomethylation (n=9) 3.09±0.11% Chronic lymphocytic leukaemia Normal (CD19+ve B cells) 4.02% Hypomethylation level cor”
- CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer (2006, methodology): “Genomic DNA was treated with sodium bisulfite and subsequently analyzed by MethyLight as describe”