A whole-genome mouse BAC microarray with 1-Mb resolution for analysis of DNA copy number changes by array comparative genomic hybridization - PubMed (original) (raw)

Comparative Study

doi: 10.1101/gr.1878804.

Jos Jonkers, Hannah Kitson, Heike Fiegler, Sean Humphray, Carol Scott, Sarah Hunt, Yuejin Yu, Ichiko Nishijima, Arno Velds, Henne Holstege, Nigel Carter, Allan Bradley

Affiliations

Comparative Study

A whole-genome mouse BAC microarray with 1-Mb resolution for analysis of DNA copy number changes by array comparative genomic hybridization

Yeun-Jun Chung et al. Genome Res. 2004 Jan.

Abstract

Microarray-based comparative genomic hybridization (CGH) has become a powerful method for the genome-wide detection of chromosomal imbalances. Although BAC microarrays have been used for mouse CGH studies, the resolving power of these analyses was limited because high-density whole-genome mouse BAC microarrays were not available. We therefore developed a mouse BAC microarray containing 2803 unique BAC clones from mouse genomic libraries at 1-Mb intervals. For the general amplification of BAC clone DNA prior to spotting, we designed a set of three novel degenerate oligonucleotide-primed (DOP) PCR primers that preferentially amplify mouse genomic sequences while minimizing unwanted amplification of contaminating Escherichia coli DNA. The resulting 3K mouse BAC microarrays reproducibly identified DNA copy number alterations in cell lines and primary tumors, such as single-copy deletions, regional amplifications, and aneuploidy.

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Figures

Figure 1

Figure 1

Distribution of spacing of the 2825 BACs used for the 3K mouse BAC microarray. The average spacing of the BACs was 0.89 Mb across the 19 autosomes and chromosome X. Linear map positions and other details of the 3K mouse BAC clone set are available online from the Ensembl database (http://www.ensembl.org/Mus\_musculus/cytoview).

Figure 2

Figure 2

Detection of single-copy differences. (A) Normal vs. normal hybridization of spleen DNA from a C57BL/6J female. (B) Comparative genomic hybridization of normal C57BL/6J female DNA vs. normal C57BL/6J male DNA. Plotted are log2-transformed hybridization ratios against the linear map position of the BACs (in Mb). Confidence levels were calculated according to the Rosetta Error Model. Red indicates significant gain in DNA copy number, green indicates significant loss, and black indicates no significant change. The CGH profile of the female vs. male hybridization shows identical DNA copy numbers for all autosomes (mean log2 ratio -0.0066, s.d. of the log2 ratios = 0.064); the copy numbers of male and female chromosome X sequences were clearly different (mean log2 ratio of 0.63).

Figure 3

Figure 3

Detection of regional single-copy deletions. (A) Comparative genomic hybridization of normal AB2.2 ES cell DNA vs. DNA from 7A9 ES cells, harboring a regional single-copy deletion between D4Mit117 (94.6 Mb) and D4Mit246 (96.6 Mb) on chromosome 4. (B) Comparative genomic hybridization of normal AB2.2 ES cell DNA vs. DNA from 3D5 ES cells, harboring a 6.9-Mb single-copy deletion from Mpo2 (88.5 Mb) to Chad2 (95.4 Mb) on chromosome 11. The BAC clones spanning the deletion regions (green dots) are denoted. Both deletions were confirmed by FISH analysis. On prometaphase chromosomes from 7A9 and 3D5 ES cells, only one hybridization signal was observed, with probes corresponding to the deleted region (red signals, indicated by red arrow), whereas two hybridization signals, derived from both alleles, were detected with probes located outside of the deletion regions (green signals, indicated by green arrows).

Figure 4

Figure 4

Array CGH analysis of oncogene amplifications in mouse mammary tumors. (A) Southern analysis of tumor DNA using Kras2, Myc, and Ccnd1 specific probes. Tumors 1, 2, and 3 showed amplification of Kras2, Myc, and Ccnd1, respectively. (B) Whole-genome array CGH profiles (left panels) and CGH profiles of the relevant chromosomes (right panels) from mouse mammary tumors 1, 2, and 3. Plotted are log2-transformed hybridization ratios of tumor DNA vs. control DNA. Vertical red bars on each chromosome indicate the positions of Kras2, Myc, and Ccnd1, respectively.

Figure 4

Figure 4

Array CGH analysis of oncogene amplifications in mouse mammary tumors. (A) Southern analysis of tumor DNA using Kras2, Myc, and Ccnd1 specific probes. Tumors 1, 2, and 3 showed amplification of Kras2, Myc, and Ccnd1, respectively. (B) Whole-genome array CGH profiles (left panels) and CGH profiles of the relevant chromosomes (right panels) from mouse mammary tumors 1, 2, and 3. Plotted are log2-transformed hybridization ratios of tumor DNA vs. control DNA. Vertical red bars on each chromosome indicate the positions of Kras2, Myc, and Ccnd1, respectively.

Figure 5

Figure 5

Array CGH analysis of in vitro transformed RMT18 MEFs derived from a female embryo. (A) CGH profiles of all autosomes and the X-chromosome, showing copy number gains for chromosomes 6, 10, and 19, and copy number loss for chromosome X. (B) Ploidy analysis of RMT18 cells. Normal mouse ES cells were used as a normal diploid control. DNA index is 1.37. (_C_-E) Chromosome painting of RMT18 metaphase spreads for chromosome 19, X, and 18, respectively.

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WEB SITE REFERENCES

    1. http://www.ensembl.org/mus_musculus/cytoview; map positions of 1-Mb mouse BAC clone set.
    1. http://www.sanger.ac.uk/Software/Image; fingerprint gel image analysis software.
    1. http://www.bcgsc.ca/lab/mapping/mouse; mouse BAC fingerprints reference database.
    1. http://www.sanger.ac.uk/Projects/Microarrays/arraylab/protocol4; microarray spotting and post-spotting processing protocols.
    1. http://download.cell.com/supplementarydata/cell/102/1/109/DC1/ErModlv2.htm; Rosetta error model.

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