16S Ribosomal Dna Amplification For Phylogenetic Study

OF BACTERIOLOGY, Jan. 1991, p. 697-703 0021-9193/91/020697-07$02.00/0 Copyright © 1991, American Society for Microbiology

JOURNAL

Vol. 173, No. 2

16S Ribosomal DNA Amplification for Phylogenetic Study
WILLIAM G. WEISBURG,* SUSAN M. BARNS, DALE A. PELLETIER, AND DAVID J. LANE GENE-TRAK Systems, 31 New York Avenue, Framingham, Massachusetts 01701

Received 16 April 1990/Accepted 7November 1990

A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful forvarious exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition,16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can bederived from a readily obtainable lyophilized bacterial culture.
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The comparison of rRNA sequences is a powerful tool for deducing phylogenetic and evolutionary relationships among bacteria, archaebacteria, and eucaryotic organisms. These sequences have been derived previously by methods including oligonucleotide cataloging (6), sequencingof clones, direct sequencing of RNA by using reverse transcriptase (11), and sequencing of material amplified by polymerase chain reaction (PCR) (3, 5, 15). The present study expands on the use of DNA amplification technology for the study of rRNA sequences within the eubacteria. Several primers are described, and novel methods for their use are presented. Sogin and coworkers (15) described aprimer pair for the enzymatic amplification of eucaryotic small-subunit rRNA gene (rDNA) sequences. Boettger (3) and Edwards and coworkers (5) examined the sequence of Mycobacterium bovis 16S rRNA PCR products without cloning. They also examined a limited set of organisms with their PCR primers: four gamma purple bacteria and two gram-positive species with high G+C content were experimentallyamplified. The present study describes primers for the amplification of most eubacterial 16S rRNAs, details preparation of samples, and presents our current sequencing procedures.
MATERIALS AND METHODS Nucleic acid preparation. Genomic DNAs were prepared by using standard methods (13, 14). Preparation of DNA from lyophilized ampoules consisted of suspending the lyophilized cells in 0.2 ml of 10 mM Trishydrochloride (pH 8.3)-2.5 mM MgCl2-50 mM KCl. This was added to approximately 0.1 ml of 0.1-mm-diameter acid-washed glass beads, 10 ,u of 20% sodium dodecyl sulfate, and approximately 200 RI of phenol, saturated with the above buffer. This sample was shaken in a 500-,u microcentrifuge tube, seated within a 2-ml tube, in a Mini-Bead Beater (Biospec Products) for 2 min. After phase separation (10min at approximately 15,000 x g), the aqueous phase was ethanol precipitated. The pellet was suspended in 20 to 50 RI of TE buffer (10 mM Tris hydrochloride [pH 8.0], 1 mM EDTA), and between 2 and
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Corresponding author.
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5% of this resuspended DNA was put into the PCR amplification. PCR amplification and purification of product. Approximately 1 to 3 ,ug of genomic DNA was amplified in...