ACCURATE INSERTIONAL INACTIVATION OF LACZ-ALPHA - CONSTRUCTION OF PTRUEBLUE AND M13TRUEBLUE CLONING VECTORS

Citation
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
Citations number
23
Categorie Soggetti
Genetics & Heredity
Journal title
GeneACNP
ISSN journal
03781119
Volume
213
Issue
1-2
Year of publication
1998
Pages
83 - 91
Database
ISI
SICI code
0378-1119(1998)213:1-2<83:AIIOL->2.0.ZU;2-H
Abstract
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.