Technical demonstration of whole genome array comparative genomic hybridization.
Array comparative genomic hybridization (array CGH) is a method for detecting gains and losses of DNA segments or gene dosage in the genome. 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. 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 approximately 100 kilobase pair (kb) segments. These BAC targets are individually synthesized and spotted in duplicate on a single glass slide. 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. The SMRT array can detect alterations as small as 50 kb in size. 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 (25-100 ng) this allows profiling of precious samples such as those produced by microdissection. 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. 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.
Kennett
Jennifer Y
JY
Department of Cancer Genetics, BC Cancer Research Centre. jkennet@bccrc.ca
Watson
Spencer K
SK
Saprunoff
Heather
H
Heryet
Cameron
C
Lam
Wan L
WL
D002232:Carbocyanines / Q000737:chemistry; D022202:Chromosomes, Artificial, Bacterial; D055028:Comparative Genomic Hybridization / Q000379:methods*; D004247:DNA / Q000032:analysis / Q000235:genetics; D015894:Genome, Human; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*
D002232:Carbocyanines; C089187:cyanine dye 3; C085321:cyanine dye 5; D004247:DNA