( hybridized with a human b-actin cDNA p...)Human Universal cDNA Library Array I

An aliquot of this pooled cDNA was used to construct a primary library. The cDNA was annealed with the adapter 5-(A)₁₈CCCGGGAGCT-3, and the plasmid pT7T3 18 U that had been linearized with Pst I, (dC)-tailed and digested with Sac I. Once the single-stranded region was ligated and repaired, it produced recombinant molecules in which the inserts were bound at their 5 termini by a poly(C) and at their 3 termini by an Sac I site (reconstituted by the cohesive ends of the primer/adapter and vector). These molecules were transformed into E. coli resulting in 2 x 10⁵ ampicillin-resistant clones.

Figure 1

The remaining cDNA was size-fractionated by electrophoresis in a low melting point agarose gel. One-third of the total cDNA was used to select molecules longer than 1 kb, while two-thirds was used to select for molecules longer than 2 kb. The two resulting fractions were combined to obtain a population enriched for high molecular weight cDNAs without losing representation of the smaller full-length molecules (Figure 1).

The cDNA obtained by this fractionation procedure was used in the construction of two normalized libraries.³ The corresponding mRNAs used for cDNA synthesis were pooled, biotinylated with photobiotin, and hybridized in excess with the cDNA. Streptavidin treatment, followed by phenol extraction, removed cDNA-mRNA hybrids and mRNA so that only the unhybridized cDNA remained in the aqueous phase. One-half of this unhybridized cDNA was used to construct a library of 2 x 10⁵ clones (as p
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