Accurate insertional inactivation of lacZα: construction of pTrueBlue and M13TrueBlue cloning vectors

Color selection plasmid and phage vectors based on insertional inactivation of the lacZα gene fragment have been in wide use for nearly two decades. The originals (the pUC and M13mp series) and all subsequent derivatives of these vectors contain the same mechanism for insertional inactivation of lacZα placing the sites for gene interruption between the initiator ATG codon and codon 7, a region that encodes a functionally nonessential part of the lacZα-complementation peptide. Numerous observations found in the literature and made in our laboratory suggest that the color selection process in these vectors does not function reliably and that erroneous conclusions about the success of cloning experiments are frequently drawn. We have studied the efficiency of lacZα inactivation by performing insertions at different intervals along the entire length of the proximal 60-amino-acid (aa)-coding region of the lacZ gene ( lacZα). Our results show that accurate and reliable inactivation of lacZα occurs only when insertions are made within the DNA region that encodes aa 11–36. Based on these results, novel color selection plasmid and phage vectors, pTrueBlue and M13TrueBlue, have been constructed. Application of these vectors for gap-free shotgun sequencing of genomic DNA is discussed.