PCR-based preparation of 23S rRNA-targeted group-specific polynucleotide probes.
DNA coding for a variable region within domain III of bacterial 23S rRNA was used as the target for group-specific polynucleotide hybridization probes. The corresponding rDNA was amplified in vitro by the PCR technique in combination with a pair of primers specific for flanking conserved target sites. The amplified fragments were cloned or used directly as probes. RNA probes were generated by in vitro transcription of cloned or amplified rDNA. The probes were labeled by incorporating modified nucleotides during in vitro DNA amplification or in vitro transcription or by random priming. The use of in vitro transcribed single-stranded RNA probes instead of double-stranded DNA probes provided stronger hybridization signals. Group-specific probes were prepared from genomic DNAs or directly from cells of Acinetobacter calcoaceticus, Alcaligenes faecalis, Aeromonas hydrophila, Nannocystis exedens, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Pseudomonas stutzeri.
Ludwig
W
W
Lehrstuhl für Mikrobiologie, Technische Universität München, Federal Republic of Germany.
Dorn
S
S
Springer
N
N
Kirchhof
G
G
Schleifer
K H
KH
D001419:Bacteria / Q000145:classification / Q000235:genetics; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000138:chemical synthesis / Q000235:genetics*; D004275:DNA, Ribosomal / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D015342:DNA Probes; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S