Sn. Slilaty et S. Lebel, ACCURATE INSERTIONAL INACTIVATION OF LACZ-ALPHA - CONSTRUCTION OF PTRUEBLUE AND M13TRUEBLUE CLONING VECTORS, Gene, 213(1-2), 1998, pp. 83-91
Color selection plasmid and phage Vectors based on insertional inactiv
ation of the lacZ alpha gene fragment have been in wide use for nearly
two decades. The originals (the pUC and M13mp series) and all subsequ
ent derivatives of these vectors contain the same mechanism for insert
ional inactivation of lacZ alpha placing the sites for gene interrupti
on between the initiator ATG codon and codon 7, a region that encodes
a functionally nonessential part of the lacZ alpha-complementation pep
tide. Numerous observations found in the literature and made in our la
boratory suggest that the color selection process in these vectors doe
s not function reliably and that erroneous conclusions about the succe
ss of cloning experiments are frequently drawn. We have studied the ef
ficiency of lacZa inactivation by performing insertions at different i
ntervals along the entire length of the proximal 60-amino-acid (aa)-co
ding region of the lacZ gene (lacZ alpha). Our results show that accur
ate and reliable inactivation of lacZ alpha occurs only when insertion
s are made within the DNA region that encodes aa 11-36. Based on these
results, novel color selection plasmid and phage vectors, pTrueBlue a
nd M13TrueBlue, have been constructed. Application of these vectors fo
r gap-free shotgun sequencing of genomic DNA is discussed. (C) 1998 El
sevier Science B.V. All rights reserved.