Accurate insertional inactivation of lacZalpha: construction of pTrueBlue and M13TrueBlue cloning vectors.
Color selection plasmid and phage vectors based on insertional inactivation of the lacZalpha 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 lacZalpha placing the sites for gene interruption between the initiator ATG codon and codon 7, a region that encodes a functionally nonessential part of the lacZalpha-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 lacZalpha inactivation by performing insertions at different intervals along the entire length of the proximal 60-amino-acid (aa)-coding region of the lacZ gene (lacZalpha). Our results show that accurate and reliable inactivation of lacZalpha 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.
Slilaty
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Genomics One Corporation, 3030 Boulevard Le Carrefour, Suite 1004, Laval, Quebec, H7T 2P5, Canada. slilaty@genomicsone.com
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D002863:Chromogenic Compounds / Q000378:metabolism; D003001:Cloning, Molecular; D004274:DNA, Recombinant / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D016368:Frameshift Mutation; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics*; D007763:Lac Operon; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017422:Sequence Analysis, DNA; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D001426:Bacterial Proteins; D002863:Chromogenic Compounds; D004274:DNA, Recombinant; D001616:beta-Galactosidase