Rapid genome walking: a simplified oligo-cassette mediated polymerase chain reaction using a single genome-specific primer.
In the present report we show that unknown DNA fragments are easily amplified in a single PCR reaction from an oligo-cassette library with a single genome-specific primer in combination with a cassette-specific primer. The novelty of the system, in comparison to the vectorette PCR method, lies in the use of unphosphorylated in contrast with phosphorylated oligo-cassettes in the ligation to the chromosomal DNA fragments. After denaturation of the DNA library, all chromosomal fragments carry a single-stranded linker attached to the 5'-end only. Therefore, the presence of the vectorette mismatched region is not required when unphosphorylated cassettes are used. As an example we report the amplification of the era gene from Lactococcus lactis.
Kilstrup
M
M
Department of Microbiology, Technical University of Denmark, Building 301, DK2800 Lyngby, Denmark. immk@pop.dtu.dk
Kristiansen
K N
KN
D001426:Bacterial Proteins / Q000235:genetics; D016386:Chromosome Walking / Q000379:methods*; D002876:Chromosomes, Bacterial; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics*; D029968:Escherichia coli Proteins; D019204:GTP-Binding Proteins / Q000235:genetics; D015723:Gene Library; D016678:Genome; D013294:Lactococcus lactis / Q000235:genetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D016133:Polymerase Chain Reaction / Q000379:methods*; D016601:RNA-Binding Proteins
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D016601:RNA-Binding Proteins; C068027:era protein, E coli; D019204:GTP-Binding Proteins