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1Department of Cancer Genetics, BC Cancer Research Centre, 2Deeley Research Centre, BC Cancer Agency, 3Photography/Video Production, Multi-Media Services, BC Cancer Agency
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*The slides can remain in the
This video is a technical demonstration of the hybridization protocol for whole genome tiling path array CGH, which scans the entire human genome using only 25-100 ng of DNA that can be isolated from a variety of sources, including archival formalin fixed material.
Kennett, J. Y., Watson, S. K., Saprunoff, H., Heryet, C., Lam, W. L. Technical Demonstration of Whole Genome Array Comparative Genomic Hybridization. J. Vis. Exp. (18), e870, doi:10.3791/870 (2008).
Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome 1. Recent advances in this technology have enabled high resolution comparison of whole genomes for the identification of genetic alterations in cancer and other genetic diseases 2. The Sub-Megabase Resolution Tiling-set array (or SMRT) array is comprised of a set of approximately thirty thousand overlapping bacterial artificial chromosome (BAC) clones that span the human genome in ~100 kilobase pair (kb) segments 2. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide 2-4. Array CGH is based on the principle of competitive hybridization. Sample and reference DNA are differentially labeled with Cyanine-3 and Cyanine-5 fluorescent dyes, and co-hybridized to the array. After an incubation period the unbound samples are washed from the slide and the array is imaged. A freely available custom software package called SeeGH (www.flintbox.ca) is used to process the large volume of data collected - a single experiment generates 53,892 data points. SeeGH visualizes the log2 signal intensity ratio between the 2 samples at each BAC target which is vertically aligned with chromosomal position 5,6. The SMRT array can detect alterations as small as 50 kb in size 7. The SMRT array can detect a variety of DNA rearrangement events including DNA gains, losses, amplifications and homozygous deletions. A unique advantage of the SMRT array is that one can use DNA isolated from formalin fixed paraffin embedded samples. When combined with the low input requirements of unamplified DNA (25-100ng) this allows profiling of precious samples such as those produced by microdissection 7,8. This is attributed to the large size of each BAC hybridization target that allows the binding of sufficient labeled samples to produce signals for detection. Another advantage of this platform is the tolerance of tissue heterogeneity, decreasing the need for tedious tissue microdissection 8. This video protocol is a step-by-step tutorial from labeling the input DNA through to signal acquisition for the whole genome tiling path SMRT array.
Note: limit exposure of Cye Dyes to light at all times (this can be achieved by working in a darkened area or by shielding the tubes with a cover such as aluminum foil)
SAMPLE CLEAN UP
(Combined probe clean-up and preparation for hybridization)
CALCULATION OF INCORPORATIONS
Removal of the slide from the hybridization cassette is critical. DIG Easy quickly crystallizes at room temperature. The slide should be immediately immersed and the cover slip removed in the wash solution.
Poor quality DNA will not provide a good hybridization profile. It is essential to ensure that sample and reference DNA are free from contaminants such as phenol, RNA, salt, etc that may interfere with the random prime labeling step before starting a hybridization experiment. For example the resuspension of DNA in Tris - EDTA (TE) instead of water is not recommended as high salt concentration can inhibit the labeling reaction. We recommend assaying DNA quality using the Nanodrop spectrophotometer to measure both the DNA quantity as well as overall shape of absorbance curve and the 260/280, 260/230 ratio.
Washing: Removing the slide from the hybridization cassette and transferring to the heated wash solution is a critical step in the hybridization process as the DIG Easy hybridization solution crystallizes very quickly. It is important that the wash solutions be at a neutral pH and the temperature of the wash solution is important for stringency while removing the unbound probe. After drying slides it is important to keep them in the dark if not scanning right away, or after scanning if you wish to rescan them at a later time.
Ozone: Ozone has been a worldwide concern when performing array CGH. Ozone levels above 5ppm have been shown to adversely affect the Cye dyes. Cye 5 is particularly sensitive to ozone degradation. Minimizing ozone exposure is key to preventing Cye dye degradation and loss of signal before and during scanning. Scanning at night for example, when environmental ozone is typically lower, is one possible option. Ozone free enclosures for automated slide washing and scanning and various chemical slide treatments are commercially available. Ozone resistant Cye dyes have also recently been developed.
We wish to thank members of the Wan Lam Lab BAC Array team especially Miwa Suzuki and Bryan Chi for the preparation of this article. This work was supported by funds from Canadian Institutes for Health Research, Genome Canada/Genome British Columbia, and NIH/NIDCR grant RO1 DE15965-01.
|5X Klenow Buffer||Reagent||Promega Corp.|
|Random Octamers||Reagent||Alpha DNA|
|10X dNTP mix||Reagent||Promega Corp.||2mM dATP, dGTP, dTTP, 1.2mM dCTP|
|Cy-3 labeled dCTP||Reagent||GE Healthcare|
|Cy-5 labeled dCTP||Reagent||GE Healthcare|
|Human Genomic DNA Reference||Reagent||Novagen, EMD Millipore||Example of a possible reference|
|YM-30 Column||Reagent||EMD Millipore|
|DIG Easy||Reagent||Roche Group|
|Sheared Herring Sperm DNA||Reagent||Promega Corp.|
|Coverslip||Reagent||Fisher Scientific||22mm x 60mm|
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