The case of chicken attachment region binding protein reinforces the importance of pre-screening of several libraries for cDNA cloning

cDNA cloning very often involves screening of a library by hybridization with a short fragment of the desired cDNA as a probe. Commonly the choice of the library is made on the basis of the properties of the vector, the extent of expression in the tissue from which the library is made, the `quality' of the library, and possibly other parameters. However, even when these choices are optimally made, several caveats have to be considered. For example, a high expression rate is not a guarantee that a corresponding high number of positive clones will be obtained, and cases have been reported of successful screening of a library prepared from a weakly expressing tissue. Furthermore, particular mRNAs are difficult to reverse-transcribe, possibly owing to a high CG content or base-paired structures, leading to an under-representation of the desired cDNA clone in the library, CG-rich sequences generally behave problematically in polymerase reactions, even at elevated temperatures (72 degree C), and several attempts have been made to solve these problems. In the severe case reported here, we attempted to clone the cDNA of the chicken nuclear-matrix protein ARBP (attachment region binding protein). We first synthesized a single-stranded cDNA by reverse transcription of chicken brain polyadenylated RNA and produced a 200 bp fragment by PCR amplification using oligonucleotide primers derived from partial peptide sequences of the purified protein. In a long-lasting screening project employing this fragment as a hybridization probe, we were unable to find any positive clone among a million phages each from a lambda gt 10 brain library (Clontech), a \g?\ZAP library from MSB-1 cells, or a lambda ZAP liver library (Stratagene). We therefore decided to use a second PCR amplification that generates a 107 bp fragment as a pre-screening analysis to evaluate several alternative libraries for the presence of ARBP cDNA inserts. As differential decreases in phage titre would lead to erroneous results, we first amplified these libraries.