Rapid 16S ribosomal DNA sequencing from a single colony without DNA extraction or purification.
Ribosomal RNA sequences are useful for establishing phylogenetic relationships, for oligonucleotide probes and for characterization of uncultured organisms. We describe rapid ribosomal DNA sequencing using PCR with transcript sequencing. Nucleic acid specificity at three steps (amplification, transcription and sequencing) eliminated the need for nucleic acid extraction or purification. Sequence was obtained from a crude lysate from a single colony of bacteria. The basic sequencing method should be adaptable to provide rapid sequence information in a wide variety of applications.
Frothingham
R
R
Department of Medicine, Duke Univ. Medical Center, Durham, NC 27710.
Allen
R L
RL
Wilson
K H
KH
D001345:Autoradiography; D001483:Base Sequence; D004269:DNA, Bacterial / Q000302:isolation & purification; D004275:DNA, Ribosomal; D003854:Deoxyribonucleotides; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D003854:Deoxyribonucleotides; D012336:RNA, Ribosomal, 16S; D012194:RNA-Directed DNA Polymerase