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.
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