A tiling resolution DNA microarray with complete coverage of the human genome

Abstract

We constructed a tiling resolution array consisting of 32,433 overlapping BAC clones covering the entire human genome. This increases our ability to identify genetic alterations and their boundaries throughout the genome in a single comparative genomic hybridization (CGH) experiment. At this tiling resolution, we identified minute DNA alterations not previously reported. These alterations include microamplifications and deletions containing oncogenes, tumor-suppressor genes and new genes that may be associated with multiple tumor types. Our findings show the need to move beyond conventional marker-based genome comparison approaches, that rely on inference of continuity between interval markers. Our submegabase resolution tiling set for array CGH (SMRT array) allows comprehensive assessment of genomic integrity and thereby the identification of new genes associated with disease.Identification of chromosomal imbalances and variation in DNA copy-number is essential to our understanding of disease mechanisms and pathogenesis. Array CGH 1 or matrix CGH 2 offers the highest resolution for practical genome-wide detection of chromosomal alterations. This technique is derived from the concept of conventional CGH 3 , which has contributed greatly to the molecular characterization of both somatic and constitutional genomic DNA mutations over the last decade 4-6 . The primary limitation of conventional CGH is its resolution (∼20 Mb), as this method detects segmental copy-number changes on metaphase chromosomes 3 . In array CGH, the metaphase chromosome spread is replaced by BACs, PACs or YACs containing human DNA as targets, increasing the resolution to the distance between the selected marker DNA clones 1,2 . Genome screening using array CGH has great potential in the characterization of numerous chromosomal disorders.Efforts to construct DNA arrays spanning the human genome consisted of spotting 2,460 (ref. 7) or 3,500 (ref. 8) marker BAC clones representing the sequenced genome at an average interval of ∼1 Mb.These studies showed that sufficient target-DNA printing solution could be generated from individual BACs using PCR-based protocols. Because the target product is PCR-derived, it is easily replenishable, obviating the need for multiple rounds of laborious large-scale BAC DNA preparations. These arrays are sensitive enough to detect singlecopy changes, but the technique is limited by the small number of BAC markers representing the genome on the slide, rather than the methodology. Even at this resolution, array CGH is useful for detecting chromosomal aberrations associated with congenital abnormalities and somatic malignancies [9][10][11][12] .Recent studies focused on higher-density regional arrays for fine mapping and identifying new genes in specific chromosomal regions [13][14][15][16][17][18] . For example, a candidate oncogene for association with

Genome-wide screening for genetic changes in a matched pair of benign and prostate cancer cell lines using array CGH

“…aCGH also showed a gain of the q-arm of chromosome 20 in the cancer cell line CP 3 Genetic changes in prostate cancer cells N Brookman-Amissah et al common normal reference DNA in studies of this kind. The use of more sensitive 'tiling path' arrays, which cover the genome at a much higher resolution, and hence map alterations to more discrete regions, 26 will enable better identification of chromosomal changes that are critical for the onset of prostate cancer and its progression, profiling of prostate tumours, and the eventual possible identification of markers associated with the outcome of treatment. Genetic changes in prostate cancer cells N Brookman-Amissah et al…”

Genome-wide analysis of allelic imbalance in prostate cancer using the Affymetrix 50K SNP mapping array

“…Until recently, the technology represented by PCR-based determination of microsatellites only allowed a modest number of polymorphic markers to be used, which limited the resolution of the technique. With the development of comparative genomic hybridisation (CGH) arrays using more than 30 000 BAC clones spanning the human genome (Ishkanian et al, 2004), and highdensity single-nucleotide polymorphism (SNP) microarrays designed to genotype more than 100 000 SNPs in the human genome DNA (Matsuzaki et al, 2004), the resolution of the whole genome scanning technique has increased considerably and allowed accurate and reproducible determination of copy number changes in the cancer genome (Goyama et al, 2004;Nannya et al, 2005). The SNP array offers the possibility to analyse LOH and generate accurate copy number simultaneously in a high-throughput and high-resolution genome-wide manner, thus making it possible to distinguish between LOH regions with underlying hemizygous deletions and those with copy-neutral events (Zhao et al, 2004).…”

Array-based comparative genomic hybridization of mapped BAC DNA clones to screen for chromosome 14 copy number abnormalities in meningiomas

“…27,28 To the best of our knowledge, this is the first report in which high-resolution a-CGH is applied to the analysis of chromosome 14 in meningiomas. The increased resolution of a-CGH is directly related to the development of thousands of BAC clones that allow for the analysis of the entire human genome including chromosome 14 23 and the availability of overlapping BAC clones for each chromosome region of interest. Adjacent and overlapping clones can be used together to increase the efficiency and accuracy of the analysis, as the tile-path array in which a set of overlapping clones are used, deletions or gains larger than or equal to a single clone, frequently involve also neighbouring clones.…”