Limitations on the recombinant plasmid selection by Lac+/Lac colony phenotype detection

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Abstract

Lac+/Lac selection of recombinant plasmids based on the insertional inactivation of LacZα gene cannot differentiate recombinant clones in some cases. Several fragments of exon 11 of human brca1 gene were cloned in LacZα-containing plasmids so that frameshift appeared at the 5-end of the fragments tested but these fragments were in frame with the part of LacZα situated downstream of the polylinker. All plasmids except one caused blue colonies formation after being transformed in Escherichia coli LacZΔM15 cells in spite of the frameshift. The fact may be explained by reinitiation of translation within the mRNA transcribed from the inserted DNA fragments at in-frame AUG, GUG, and UUG. The data demonstrated limitations on the Lac+/Lac selection of LacZα-based recombinant plasmids.

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Materials and methods

Enzymes and chemicals. Taq DNA polymerase, T4 DNA ligase, dNTPs, and the kit for DNA isolation were from “MedigenLab” (Novosibirsk, Russia). Restriction endonucleases, T4 polynucleotide kinase, and plasmid pUC19 were from “SibEnzyme” (Novosibirsk, Russia). Plasmids pGEM7Zf and pBluescript IISK were from “Promega” (USA) and Stratagene (USA), respectively. Guanidine thiocyanate, calcium chloride, peptone, yeast extract, and BactoAgar were from “Fluka” (Switzerland). Silica (Silicon dioxide) and o

Results and discussion

A large part of exon 11 of human brca1 gene (2.97 kb, EcoRI/AsuNHI) was cloned in pGEM7Zf plasmid and the resultant plasmid was designated as pGEM7f+ex11. The cloning was designed in-frame with LacZα gene. All colonies of XL-1 Blue E. coli cells with the recombinant plasmid were blue on the indicator plates in spite of the proved 2.97 kb DNA insert. The activity of β-galactosidase in E. coli cells with the recombinant plasmid (954±67Millerunits) did not differ from β-galactosidase activity of the

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