A single oligonucleotide can be used to rapidly isolate DNA sequences flanking a transposon Tn5 insertion by the polymerase chain reaction.
We have developed a strategy to rapidly construct DNA hybridization probes for the isolation of genes disrupted by transposon Tn5 insertions. A single oligonucleotide complementary to and extending outward from the ends of the inverted repeat of Tn5 was used to prime DNA synthesis in the polymerase chain reaction. The amplified product consisted of DNA sequences adjacent to both ends of the transposon insertion. The general feasibility of the approach was tested by amplifying pBR322 sequences from a derivative of pBR322 containing a Tn5 insertion. To amplify genomic DNA sequences flanking a Tn5 insertion in the chromosome of a Pseudomonas syringae strain, circular substrates were generated by ligating EcoRI-digested genomic DNA. Tn5 was contained intact within one such circular molecule, as the transposon does not contain sites for cleavage by EcoRI. The amplified product (approximately 2.5 kb) was used as a DNA hybridization probe to isolate the homologous fragment from a cosmid library of wild-type Pseudomonas syringae genomic DNA. This approach may be applied to the efficient isolation of sequences flanking any Tn5 insertion.
Rich
J J
JJ
Department of Plant Pathology, University of Wisconsin, Madison 53706.
Willis
D K
DK
D001483:Base Sequence; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000737:chemistry*; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011549:Pseudomonas / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D009841:Oligonucleotides