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DNA Microarray Analysis
The following DNA microarray services are provided by the facility. Please order online or contact Jeff Conroy for details.
- DNA preparation (fresh, frozen, paraffin embedded)
- DNA amplification and QC
- Probe generation
- 21K Human BAC microarray comparative genomic hybridization (CGH array)
- Illumina Infinium BeadChip for Genotyping, CNV and LOH
- Affymetrix 6.0 SNP mapping arrays
- Slides scanning
- Image analysis and basic microarray data analysis
- BAC clone distribution for DNA probes and FISH validation
- Verification of copy number aberrations
Illumina Infinium BeadChips:
The Infinium HD products include the HumanCytoSNP-12, Human660W-Quad, HumanOmni1-Quad and Human1M-Duo BeadChips. Combined with the existing Infinium II product—HumanExon510S-Duo—Illumina BeadChips provide a broad spectrum of whole-genome DNA Analysis products to support a variety of experimental designs. Researchers have the flexibility to use panels of 370,000 to nearly 1,200,000 markers per sample, depending on their study goals. All of these BeadChips provide powerful and integrated genome-wide SNP genotyping, LOH and CNV detection.
RPCI BAC DNA Arrays:
Mouse 6.5K RPCI-23/-24 BAC array
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 ~21,000 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 150 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 Telechem pins and a MicroGrid ll TAS arrayer (DigiLab) in duplicate to create arrays up to 42,000 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 (DigiLab). After hybridization, the slides are automatically washed in a Little Dipper Ozone-free workstation 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.
Affymetrix Human SNP Array 6.0
- Unbiased selection of 482,000 SNPs comprised of historical SNPs from the SNP Array 5.0 and 500K Mapping Array Set
- Selection of additional 424,000 SNPs including; Tag SNPs, SNPs from chromosomes X and Y, Mitochondrial SNPs , New SNPs added to the dbSNP database, SNPs in recombination hotspots.
- More than 946,000 copy number probes including; 202,000 probes targeting 5,677 known CNV regions from the Toronto Database of Genomic Variants, Regions resolve into 3,182 distinct, non-overlapping segments; on average 61 probes per region, 744,000 probes, evenly spaced along the genome.
DNA Requirements
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 for the BAC arrays (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.
