Applications demonstrating DNA microarray utility

Armed with this new tool, Schena et al. (1996) created a microarray of 1,046 human cDNAs of unknown sequence. They were derived from human peripheral blood lymphocyfes fransformed wifh Epsfein-Barr virus. Suitably sized inserts [ 600 base pairs (bp)] were cloned into a lambda vector, subsequently infected into an Escherichia coli strain, and finally amplified by polymerase chain reaction (PCR) using 5 -amino-modified primers. The resulting 5 -amino-modified cDNA amplicons were then arrayed onto sily-lated microscope slides. Next, the expression levels in human Jurkat cells undergoing heat shock or phorbol ester induction were examined. 

Total mRNAs from confrol and induced cells were labeled using reverse franscripfase wifh fhe incorporation of fluorescene-dCTP (control, green label) or Cy5-dCTP (induced, red label). The two populations were hybridized to separate arrays. However, the labels were also swapped to verify fhaf any differences in labeling efficiency did not affect the result. 

Study Dlff. Expressed/Total Genes % Dlff. Expressed 

While the Schena papers (1995 and 1996) served as first demonstrations of cDNA microarray fechnology, it was clear that further refinements were necessary in order to realize the full pofenfial of the microarray. Arraying technology was in its infancy and suffered from inconsistency in uniform spotting, making it difficult to compare slides. Refinements in labeling and detection were also needed. 

The diauxic shift experiment turned out to be an extremely important demonstration. While S. cerevisiae exhibited very little in the way of differ-enhal expression achvity (19 of 6400 genes showing twofold expression) 

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