Rapid mapping of Escherichia coli::Tn5 insertion mutations by REP-Tn5 PCR.

P. S. Subramanian, J. Versalovic, E. R. McCabe, J. R. Lupski

Research output: Contribution to journalArticle

Abstract

We describe a novel method to map chromosomal Escherichia coli::Tn5 insertion mutations rapidly. This method utilizes the ends of Tn5 and the E. coli REP sequence as primer binding sites for the polymerase chain reaction (PCR). The unique E. coli chromosomal sequence located between these primer binding sites is amplified by PCR and used as a probe to identify the recombinant clones from the Kohara phage ordered E. coli miniset bank that contains the Tn5 mutated loci. We used this approach to map two Tn5 insertion mutations previously identified by their effect on glycerol metabolism. The insertion mutations mapped to glpD, the aerobic sn-glycerol-3-phosphate dehydrogenase gene. Phenotypic analysis of the mutant strains revealed one with partial GlpD activity, suggesting transposon-mediated alteration of promoter activity. This mapping method should be applicable to the rapid physical mapping of any insertion mutation in the E. coli chromosome.

Original languageEnglish (US)
Pages (from-to)187-192
Number of pages6
JournalPCR Methods and Applications
Volume1
Issue number3
StatePublished - Feb 1992
Externally publishedYes

Fingerprint

Insertional Mutagenesis
Escherichia coli
Polymerase Chain Reaction
Binding Sites
Coliphages
Glycerolphosphate Dehydrogenase
Tocopherols
Glycerol
Clone Cells
Chromosomes
Genes

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Subramanian, P. S., Versalovic, J., McCabe, E. R., & Lupski, J. R. (1992). Rapid mapping of Escherichia coli::Tn5 insertion mutations by REP-Tn5 PCR. PCR Methods and Applications, 1(3), 187-192.

Rapid mapping of Escherichia coli::Tn5 insertion mutations by REP-Tn5 PCR. / Subramanian, P. S.; Versalovic, J.; McCabe, E. R.; Lupski, J. R.

In: PCR Methods and Applications, Vol. 1, No. 3, 02.1992, p. 187-192.

Research output: Contribution to journalArticle

Subramanian, PS, Versalovic, J, McCabe, ER & Lupski, JR 1992, 'Rapid mapping of Escherichia coli::Tn5 insertion mutations by REP-Tn5 PCR.', PCR Methods and Applications, vol. 1, no. 3, pp. 187-192.
Subramanian PS, Versalovic J, McCabe ER, Lupski JR. Rapid mapping of Escherichia coli::Tn5 insertion mutations by REP-Tn5 PCR. PCR Methods and Applications. 1992 Feb;1(3):187-192.
Subramanian, P. S. ; Versalovic, J. ; McCabe, E. R. ; Lupski, J. R. / Rapid mapping of Escherichia coli::Tn5 insertion mutations by REP-Tn5 PCR. In: PCR Methods and Applications. 1992 ; Vol. 1, No. 3. pp. 187-192.
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