Human DNA has complex genomic organization, and it is characterized by large variations in DNA base composition, usually described in terms of GC content, or % GC (FIG. 1). GC is defined as a molar fraction of guanine and cytosine in a genome, molecule or DNA fragment, for example. The GC distribution and compositional heterogeneity of human DNA was initially discovered and analyzed by analytical ultracentrifugation in CsCl gradients (Guttann, T., et al., (1976); Clay, O., et al., (2003)) and later confirmed by DNA sequence analysis (Clay, O., et al., 2001; Takai, D., and Jones, P. A. (2002)]. Variations in DNA base composition are linked to variations in gene density so that the GC-rich regions are usually 10-20 times higher in genes than GC-poor regions.
The CpG dinucleotides play a very special role in human and all other mammalian organisms by providing a target for DNA methylation. DNA methylation is the post-synthetic modification that introduces a methyl group to carbon-5 of cytosine and creates 5 mC, the 5th DNA base. The CpG dinucleotides are distributed in a non-random fashion in human genomic DNA. The frequency with which CpG dinucleotides are found in a genome is much lower than expected from an average human genome G+C content, except for CpG clusters or “CpG islands” (Cross, S. H., and Bird, A. P., (1995)). The CpG islands are present in the promoter, and exonic regions of approximately 40% of mammalian genes. They vary in size from 200 bp up to 2.5 kb and constitute about 1-2% of the total human genome (see FIG. 1, a black region on the CpG distribution diagram). The average GC content of human CpG islands is about 65% (FIG. 2). but some CpG islands are extremely GC-rich and have as much as 75-80% GC content (Takai, D., and Jones, P. A., (2002)). There are about 30,000 CpG islands in the human genome, and the islands are normally unmethylated. In contrast, other regions of the genome contain few CpG dinucleotides, and these are largely methylated. Multiple findings support the idea that the transcription of genes associated with promoter CpG islands is active when these regions are in unmethylated state, and it is inhibited by promoter methylation. Methylation of promoter CpG islands plays an important role in the regulation of gene expression, development, tissue-specific gene function, genomic imprinting, and X-chromosome inactivation (see U.S. patent application Ser. No. 11/071,864 and references therein, all of which are incorporated by reference herein in their entirety). Abberant methylation patterns of CpG islands have been associated with ageing, inflammation, infectious diseases, autoimmune conditions, and carcinogenesis (see U.S. patent application Ser. No. 11/071,864 and references therein, all of which are incorporated by reference herein in their entirety).
Despite the important biological role of CpG islands and their close association with genes and gene regulation, only a few methods have been developed for purification and isolation of GC-rich DNA, and specifically, CpG islands.
Bernardi and his coworkers (Clay, O., et al., (2003)) used DNA fractionation by centrifugation in Cs2SO4 density gradients containing 3,6-bis(acetatomercurimethyl)dioxane and discovered a class of DNA with very high GC content that was particularly rich in genes and interspersed repetitive sequences.
Bird and his colleagues developed the methyl-CpG binding domain (MBD) column chromatography method (Cross, S. H., et al., (1999); Cross, S. H., et al., (2000)). In this method, DNA was digested to completion with MseI restriction enzyme, methylated at all CpGs using CpG methylase (NEB), and fractionated on a column containing Ni2+-NTA-agarose coupled with the histidine-tagged methyl-CpG binding domain protein purified from crude bacterial extracts. Eluted DNA fragments were cloned and sequenced. The method was successfully used for bulk purification and analysis of CpG islands from whole genomes (Cross, S. H., et al., (1999); Cross, S. H., et al., (2000)) and from cosmid, BAC, and PAC DNA clones (Cross, S. H., et al., (1999); Cross, S. H., et al., (2000)).
Lerman, L. S. et al., (1984) introduced the idea of analysis of DNA duplex stability using agarose gel electrophoresis of heated DNA.
Shiraishi and coworkers developed a method for preferential isolation of DNA fragments associated with CpG islands by segregation of partly melted molecules (SPM). The method is conceptually simple and uses denaturant gradient gel electrophoresis to separate DNA molecules digested with restriction endonucleases. For DNA fragments derived from the edge of CpG islands, stable partly melted molecules would be expected. When subjected to denaturing gradient gel electrophoresis, such partially melted DNA fragments are differentially retarded and retained in the gradient, while molecules with lower GC content are run off the gel. The SPM method (Shiraishi, M., et al., (1995); Shiraishi, M., et al., (1998)) and the combination of MBD column chromatography and SPM (Shiraishi, M., et al., (1998); Shiraishi, M., et al., (1999) were used to identify and isolate methylated CpG islands in human cancer cells.
Bellizzi, D., et al. (1998) used exposure of DNA fragments produced by restriction cleavage or sonication to increasing temperatures to clone thermoresistant DNA duplexes.
Although some of the methods described above have been useful for isolation, characterization and understanding the role of promoter CpG islands in normal and cancer cells, they become cumbersome in applications that involve multiple DNA samples, such as cancer diagnostics (based on analysis of promoter CpG islands hypermethylation) or high throughput DNA methylation marker discovery by hybridization to a promoter micro-array. The present invention overcomes these problems and describes a simple method for enrichment, purification, and amplification of CpG islands. The method utilizes heat treatment and enzymatic selection (rather than chromatographic methods) for isolation and in vitro amplification of GC-rich DNA and can be easily implemented in diagnostic applications and high throughput screening assays, for example.
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