An improved method for generating subtracted cDNA libraries using phage lambda vectors

We have used a biotin-based subtraction procedure to enrich a potato tuber cDNA library for sink-specific clones. The method uses single-stranded phagemids with directional inserts as both driver and target. We modified the XZAP II vector for the directional cDNA cloning and for subsequent subtractive hybridization. This improved method of subtractive hybridization circumvents many problems facing standard protocols. The present method uses the advantage of X-phage cloning which makes it possible to establish libraries from limited amounts of tissue. In vivo excision of single-stranded phagemids containing the cDNA inserts was performed to provide an unlimited source of DNA for biotinylation, hybridization and subtraction. This ability to produce unlimited quantities of DNA for subtraction is advantageous over other methods which require large amounts of RNA as the starting point (2,7,8). The phage vector system is more convenient than a plasmid/colony system (3,6,9) when the original library is to be re-screened for additional clones. The XZAP II vector was modified by introducing an adaptor (EX1: 5'-AAT 1AT CTC GAG GGC CCG ATC GGC CGA ATT CGT-3' annealed to EX2: 5'-I CGA ACG AAT TCG GCC GAT CGGGCC CTC GAG AT-3'; TIB MolBiol, Germany, Berlin) that destroys the original EcoRl and Xhol sites but contains both restriction sites in inverted orientation flanking a central Sfil site. The resulting vector was designated XPAZ II. Total RNA from sink (growing) and source (sprouting) tubers was isolated according to (4) and poly A RNA was purified by chromatography on oligo-d(T)-cellulose (Pharmacia, Type 7). cDNA libraries were constructed using Uni-ZAP cDNA synthesis system (Stratagene). The cDNAs from sink and source tubers were cloned into XZAP II and XPAZ II, respectively (Fig. 1). Both libraries had a complexity of 3 x 10 p.f.u. with -90% recombinant clones. The average size of the inserts were in the range of 1 kb. From both libraries, single stranded (ss) circular DNA were generated by in vivo excision and single-stranded DNA was isolated (6). Single-stranded DNA from source tubers was biotinylated with 1 |ig/|il long-arm Photoprobe® biotin (Vector Laboratories, Burlingame, UK; 5). Typically, 2.5 |ig ssDNA from the sink tuber library was hybridized to 20-30 (J.g of biotinylated ssDNA from source tuber, in 20 ̂ 10.75 M NaCl, 50 mM HEPES pH 7.6, 10 mM EDTA and 0.1% SDS at 65°C for 20 h under mineral oil (Sigma). Subsequently, the mineral oil was removed and the mixture was incubated with 100 |ig vectrex-avidin (Vector Laboratories) for 5 min at room temperature. Streptavidin-biotin-DNA complexes were precipitated by amsink tuber cDNA library XZAPII (sense) source tuber cDNA library XPAZII (an ti sense)