Applications of array-CGH

Recently, several applications of DNA array had been reported. The combined chromatin immuno-precipitation (ChIP) with hybridization to DNA arrays, the so-called "ChIP-on-chips" technique, has proven to be a powerful method in exploring the interaction sites of DNA-binding proteins across the entire genome.29) DNA metylation in the genome has been measured by the use of CpG island array in combination with a technique of differential methylation hybridization (DMH).30) Oligonucleotide-based microarray has been also applied to DNA methylation analysis.31,32) In addition to those, CGH-arrays have been employed for ascertaining evolutional changes in the genomes between human and non-human primates, to a complete understanding of the genetic history and evolution of human species.33)

CpG island hypermethylaton of the promoter is known to be associated with gene silencing in cancer, and now such epigenetic silencing is recognized as a 'third pathway' in Knudson's model of tumor-suppressor gene inactivation in the pathway of multi-step carcinogenesis. Several techniques have been developed to detect aberrant DNA methylation in cancer, including Methlated CpG island amplification (MCA) method.34) In order to accomplish high-throughput screening DNA metylation in the whole genome, we combined array-CGH with MCA, and our preliminary data shows that this "BAC array-based MCA (BAMCA)" can discriminate spotted BAC clones harboring DNA of methylated CpGs on an array platform as shown in Fig.3.

Fig. 3.BAMCA method
A. BAMCA: MCA method was performed as described by Toyota et al. Samples DNA of non-small cell lung cancer cell line, LK79, was digested with a methylation-sensitive restriction enzyme SmaI, followed by digested with methylation-insensitive XmaI. Adaptors are ligated to XmaI-digested sticky ends, and PCR was performed to amplify the methylation sequences in the presence of Cy3-dCTP. Control DNA is treated in the same manner with Cy5-dCTP. Closed circle indicates Methylated CpGs (marked with Me) and an open circle indicates in unmethylated CpG site.
B, the labeled samples were co-hybridized to MCG Whole Genome Array-4500. Arrays are scanned by use of a GenePix (Axon Instruments, Foster City, CA) fluorescent scanner and analyzed using ArrayGauge software (Axon Instruments). BAMCA with DNA of LK79 shows a positive BAC harboring MINT26 locus (arrow) in the partial image of the array.MINT26 has been reported as a hypermethylated locus in LK79 by Hatada I et al.37)
C, after MINT26 DNA fragments were amplified by PCR, amplicons were digested with SmaI. and electropholesed in 0.4% agarose gel. MINT26 from LK79 is digested with SmaI whereas those from lymphocytes (LC) are not digested, indicating that MINT26 locus is methylated in LK79.

This BAMCA method allows us to explore methylated CpG sites within spotted BACs and if we apply BAMCA to a tiling resolution CGH-array, methylated CpG sites emerge on an array plat form throughout the entire genome. Although across the entire human genome most cytosines within CpG dinucleotides are methylated, most investigations on the role of DNA methylation in human diseases including cancer have focused on CpG islands associated with the promoter. However, the functional elements encoded in the DNA sequence, such as transcriptional regulatory sequences other than promoters, together with determinants of chromosome structure and function remain largely unknown, because of the lack of effective and practical high throughput methods. Therefore, several applications on the array CGH platform will certainly open the door for the comprehensive identification of functional sequence elements in the human genome, facilitating us to understand sequence features that affect pathogenesis in cancer as well as unknown genetic diseases.