Human colonic biota studied by ribosomal DNA sequence analysis.
Human colonic biota is a complex microbial ecosystem that serves as a host defense. Unlike most microbial ecosystems, its composition has been studied extensively by relatively efficient culture methods. We have compared an established culture-based method with direct amplification and partial sequencing of cloned 16S rRNA genes from a human fecal specimen. Nine cycles of PCR were also compared with 35 cycles. Colonies and cloned amplicons were classified by comparing their ribosomal DNA (rDNA; DNA coding for rRNA) sequences with rDNA sequences of known phylogeny. Quantitative culture recovered 58% of the microscopic count. The 48 colonies identified gave 21 rDNA sequences; it was estimated that 72% of the rDNA sequences from the total population of culturable cells would match these 21 sampled sequences (72% coverage). Fifty 9-cycle clones gave 27 sequences and 59% coverage of cloned rDNAs. Thirty-nine rDNAs cloned after 35 cycles of PCR gave 13 sequences for 74% coverage. Thus, the representation of the ecosystem after 35 cycles of PCR was distorted and lacked diversity. However, when the number of temperature cycles was minimized, biodiversity was preserved, and there was good agreement between culturing bacteria and sampling rDNA directly.
Wilson
K H
KH
Infectious Diseases Section, VA Medical Center, Durham, NC 27705, USA. Wilso003@mc.duke.edu
Blitchington
R B
RB
D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D003001:Cloning, Molecular; D003106:Colon / Q000382:microbiology*; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000235:genetics* / Q000302:isolation & purification; D017753:Ecosystem; D005243:Feces / Q000382:microbiology; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics
D015342:DNA Probes; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S