Info

DAPI counterstain

Denaturing hybridization detection

Centromere (unlabeled)

Fig. 10.1 Comparative genomic hybridization

(

X

3

Genomic DNA sequences as targets on the microarray

Normal genomic genomic DNA DNA

Tumor

BAC/PAC/YAC

Tumor

DNA microarray

Detection

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DNA microarray

Detection

CXXXXXXXXX^^ Size

Bam H I (1) Sac II (8) Eco R I (10) Not I (11456) yASac I (20) Mlu I (22)

Mlu I (2790) Sac I(2800) Eco R I (2802) Sac II (2810) Bam H I (2811) Not I (2850)

Gain

No change

Gain

No change

Loss

Resolution

Fig. 10.2 Array comparative genomic hybridization (array CGH)

some have reported that this may be done without bias to the relative quantities of transcripts, this may not be universally true. The mRNA is reverse-transcribed, then transcribed with fluorescently labeled nucleotides to develop a fluorescently labeled complementary RNA (cRNA) representation of the original mRNA mixture. The labeled cRNA is then used for hybridization to immobilized, indexed oligonucleotide or cDNA probes. The signals at each of the loci on the slide or 'gene chip' may then be quantitated by microscopy and image analysis software. The strength of fluorescent signal may then be related to the abundance of a particular mRNA in the original sample. Comparison of different tumor samples and comparison with wild-type can then allow the determination of transcripts that are over- or under-represented in certain conditions or tumors. Tumors may be categorized using these profiles, and subgroups of messages may be used to create predictors of clinical behavior. This powerful technique has the potential to analyze an entire transcriptome of tens of thousands of genes simultaneously. It cannot determine the genomic abnormalities that lead to the differences in expression pattern, however. Given the amount of data this technique can encompass, it is possible that it will some day be a part of the routine pathological analysis of cancers, providing, like conventional pathology today, categorical and prognostic information, and possibly even directing therapeutic decision-making. The technique is currently limited in its clinical application by its relatively high cost and the relatively limited testing on tumor samples for which ample clinical details are available.

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