Arraying bacteriophage lambda libraries for screening by PCR

Abstract

▼The screening of arrayed genomic and cDNA libraries by polymerase chain reaction (PCR) is in common usage, and several libraries are readily available. In many cases the screening of such libraries by PCR allows for higher throughput compared with hybridization strategies. Examples include yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) genomic libraries, which can be screened for the locus of interest by a hierarchical series of pools, leading to a single clone. Another advantage of PCR screening is that many simple sequence length polymorphic (SSLP) loci are not suitable for screening libraries by hybridization. As it is generated, any new library can be arrayed in multiwell plates using commercially available colony/plaque-picking machines. Munroe et al. (Ref. 1) described a strategy for the screening by PCR of a cDNA library in a lambda vector, wherein 1‐2×10 6 cDNA clones were propagated as individual plaques on solid medium in 24-well culture dishes at approximately 250 plaque-forming units (pfu) per well. Phage suspensions were prepared from each well and transferred to a 96-well format. To screen the library, pools were generated that correspond to each individual 96-well plate and to each row and column within blocks of six plates each. Library screening for specific cDNA clones was conducted in a systematic and hierarchical fashion, beginning with the plate pools. However, a simple calculation reveals that approximately 4000‐8000 wells need to be plated out, which represents considerable labour. A similar strategy for screening genomic libraries was described by Israel (Ref. 2), but only 64 000 clones were screened; Sambrook et al. (Ref. 3) suggest that several hundred thousand clones of a mammalian genomic library must be screened to ensure identification of a clone for a particular locus. Both methods require additional rounds of screening to identify single positive clones. An alternative method is now presented for the arraying of lambda libraries for screening by PCR, which represents consider