MethylScreen: DNA methylation density monitoring using quantitative PCR.

Aberrant gene silencing of genes through cytosine methylation has been demonstrated during the development of many types of cancers including prostate cancer Several genes including GSTP1 have been shown to be methylated in prostate cancer leading to the suggestion and demonstration that methylation status of such genes could be used as cancer diagnosis markers alone or in support of histology. We developed a bisulfite-free alternative, MethylScreen technology, an assay for DNA methylation detection utilizing combined restriction from both methylation-sensitive restriction enzymes (MSRE) and methylation-dependent restriction enzymes (MDRE). MethylScreen was used to analyze the 5' region of GSTP1 in cell lines, in vitro methylated DNA populations, and flash-frozen tissue samples in an effort to characterize the output and analytical performance characteristics of the assay. The output from the quantitative PCR assay suggested that it could not only detect fully methylated molecules in a mixed population below the 1% level, but it could also quantify the abundance of intermediately methylated molecules. Interestingly, the interpreted output from the four quantitative PCRs closely resembled the molecular population as described by clone-based bisulfite genomic sequencing.

Comprehensive DNA methylation profiling in a human cancer genome identifies novel epigenetic targets.

Using a unique microarray platform for cytosine methylation profiling, the DNA methylation landscape of the human genome was monitored at more than 21,000 sites, including 79% of the annotated transcriptional start sites (TSS). Analysis of an oligodendroglioma derived cell line LN-18 revealed more than 4000 methylated TSS. The gene-centric analysis indicated a complex pattern of DNA methylation exists along each autosome, with a trend of increasing density approaching the telomeres. Remarkably, 2% of CpG islands (CGI) were densely methylated, and 17% had significant levels of 5 mC, whether or not they corresponded to a TSS. Substantial independent verification, obtained from 95 loci, suggested that this approach is capable of large scale detection of cytosine methylation with an accuracy approaching 90%. In addition, we detected large genomic domains that are also susceptible to DNA methylation reinforced inactivation, such as the HOX cluster on chromosome 7 (CH7). Extrapolation from the data suggests that more than 2000 genomic loci may be susceptible to methylation and associated inactivation, and most have yet to be identified. Finally, we report six new targets of epigenetic inactivation (IRX3, WNT10A, WNT6, RARalpha, BMP7 and ZGPAT). These targets displayed cell line and tumor specific differential methylation when compared with normal brain samples, suggesting they may have utility as biomarkers. Uniquely, hypermethylation of the CGI within an IRX3 exon was correlated with over-expression of IRX3 in tumor tissues and cell lines relative to normal brain samples.

Tetranucleotide frequencies in microbial genomes.

A computational strategy for determining the variability of long DNA sequences in microbial genomes is described. Composite portraits of bacterial genomes were obtained by computing tetranucleotide frequencies of sections of genomic DNA, converting the frequencies to color images and arranging the images according to their genetic position. The resulting images revealed that the tetranucleotide frequencies of genomic DNA sequences are highly conserved. Sections that were visibly different from those of the rest of the genome contained ribosomal RNA, bacteriophage, or undefined coding regions and had corresponding differences in the variances of tetranucleotide frequencies and GC content. Comparison of nine completely sequenced bacterial genomes showed that there was a nonlinear relationship between variances of the tetranucleotide frequencies and GC content, with the highest variances occurring in DNA sequences with low GC contents (less than 0.30 mol). High variances were also observed in DNA sequences having high GC contents (greater than 0.60 mol), but to a much lesser extent than DNA sequences having low GC contents. Differences in the tetranucleotide frequencies may be due to the mechanisms of intercellular genetic exchange and/or processes involved in maintaining intracellular genetic stability. Identification of sections that were different from those of the rest of the genome may provide information on the evolution and plasticity of bacterial genomes.