The following aCGH-related services are provided by the facility. Please complete an order form or contact Jeff Conroy for details.

• DNA preparation (fresh, frozen, paraffin embedded)
• DNA amplification and QC
• Probe generation
• BAC and oligonucleotide aCGH and ChIP-on-chip hybridization
• Slides scanning
• Image analysis and basic data analysis
• Advanced data analysis and statistical support
• BAC clone distribution
• Verification of copy number aberrations

Human 6K RPCI-11 BAC array

Human 19K RPCI-11 BAC array

Mouse 6.5K RPCI-23/-24 BAC array

Human and mouse 244K whole genome arrays; promoter, CpG island and ENCODE (ChIP-on-chip) arrays

BAC arrays

BAC array based CGH provides a high resolution, highly efficient means to detect, quantitate and map regions of abnormal copy number (both gains and losses) by comparing the relative efficiency with which test and reference DNA hybridize across the genome (Cowell et al., 2004; Albertson, 2003; Albertson & Pinkel, 2003; Hackett et al., 2003; Hodgson et al., 2001; Snijders et al., 2001; Veltman et al., 2003). We have developed human BAC arrays comprised of ~19,000 RPCI-11 BAC clones that have either been completely sequenced or are connected to the human sequence via STS or end sequence content, and annotated for gene content (Cowell & Nowak, 2003; Snijders et al., 2001). Included in this set are ~1300 BACs containing known tumor suppressor genes, known microdeletion syndromes, telomeric BACs, oncogenes, and genes associated with the cancer phenotype. The current human genomic arrays are produced at 160 Kilobase resolution. We also have developed a mouse BAC array comprised of 6500 RPCI-23 and RPCI-24 clones. These clones are serving as landmarks in the draft sequence integrated by virtue of sequence connections and were chosen by virtue of their STS content and association with cancer.

Generation of BAC Arrays. RPCI BAC Genomic arrays were generated by ligation-mediated PCR as described (Snijders et al., 2001; Nowak et al, 2005). Slides are printed using 10K Microspot pins and a MicroGrid ll TAS arrayer (BioRobotics) in duplicate to create arrays up to 40,000 elements on amino-silanated glass slides (Schott Nexterion type A+).

DNA Labeling and aCGH. Two control DNA pools are used for aCGH analysis. The human male control and the female control pools each contain DNA from 20 cytogenetically normal individuals. The mouse male control and female control each contain DNA from the C57BL/6J mouse. Genomic DNA from reference and test samples are fluorescently labeled by random priming and hybridized for 16 hours in a GeneTAC hybridization station (Genomic Solutions). After hybridization, the slides are automatically washed in the GeneTAC station with reducing concentrations of SSC and SDS. The aCGHs can be performed as a sex-mismatch to provide an internal hybridization control for chromosome X and Y copy number differences.

Analysis. RPCI has developed a fully automated software package (aCGHView) allowing rapid identification of copy number aberrations across the whole genome and on a chromosome by chromosome basis, based on images from our arrays.

Sample preparation. Most standard genomic DNA isolation protocols will yield DNA of high enough purity for aCGH. It is highly recommended to check genomic DNA for degradation and RNA contamination. It is critical to use purified and accurate amounts of DNA for fluorescent labeling. Low molecular weight DNA can be used (paraffin embedded, formalin fixed, degraded, etc.) but results may be more unreliable. Please contact Jeffrey Conroy (716 845-3361) for details about using low molecular weight DNA, BioScore DNA quality control or DNA samples less than 500ng.

RPCI BAC Genomic arrays are for research purposes only.