Over 145,000 human cDNA clones arrayed for nucleic acid screening
Jean-Michel Llias Leonardo de Leon Bruce Jerpseth
Stratagene has organized over 145,000 high-quality clones from a universal cDNA library that represent genes expressed in 29 different human tissues. This normalized library demonstrates a good average length of inserts, low representation of the ubiquitous b-actin gene, and high percentage of full-length clones. The bacterial colonies originally arrayed in 384 microwell plates have been grown individually on agar plates in pools of 96 clones. The DNAs obtained from the 1,536 pools have been double-spotted on nylon membranes the size of a microwell plate. Stratagene now provides these membranes for screening procedures as part of a powerful and unique cloning system that allows human genes to be fully isolated in a short period of time with only two hybridization steps.
The Human Universal cDNA Library (HUCL) was constructed essentially by a primer/adapter method.1 The cDNAs were synthesized independently from each mRNA source using a synthetic oligonucleotide, 5-CCCGGG(T)18-3, as a primer. The mRNAs were then removed by alkaline hydrolysis, and the remaining single-stranded cDNA molecules were (dG)-tailed. At this step, an aliquot of each sample was electrophoresed on an agarose gel for analysis, and the cDNAs corresponding to the 29 organs or tissues listed in Table 1 were selected and pooled.