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Stratagenes Human cDNA Microarrays: Perform Gene Expression Profiling and,,,Discover New Genes

ed by gel electrophoresis. If the cDNA template contained minor amounts of contaminating DNA, such DNA will not amplify with the insert-specific primer. Moreover, if the cDNA templates have been mixed-up in a prior step, a PCR product of the predicted length will not be amplified. Thus, PCR with an insert-specific primer both purifies and confirms the identity of the cDNA.

Fig.2

High Hybridization Specificity

Genes are often grouped into families based upon regions of protein homology and, frequently the nucleotide sequences of the protein coding regions have sufficient nucleotide homology to cross hybridize. In addition, 3 untranslated regions sometimes contain Alu sequences and other repeat elements which can cross hybridize. For these reasons, the 3 insert-specific primer was designed to amplify a portion of the cDNA including little or no coding region and excluding repeat elements. Hence, this design increases hybridization specificity when using the discovery-3 microarray by minimizing the chances that the DNA in any given spot will cross hybridize with RNA-derived probes from other gene family members or with repeat elements.

Control Spots on Discovery-3 Microarrays

Each GeneConnection discovery-3 microarray has control spots distributed throughout the microarray in a defined pattern to assist in quality control and orientation of the microarray. There are five types of control spots: spotting buffer only, human -actin DNA, and three Arabidopsis thaliana DNA. The A. thaliana genes were selected because they are involved in plant-specific processes and do not have known human homologues. In addition, they do not hybridize to membrane-arrayed human cDNA from 29 different tissues. In gene expression profiling experiments, the human -actin gene should hybridize with most, if not all, human R
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