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 be en studied extensively by relatively efficient culture methods. We hav e compared an established culture-based method with direct amplificati on and partial sequencing of cloned 16S rRNA genes from a human fecal specimen, Nine cycles of PCR were also compared with 35 cycles, Coloni es and cloned amplicons were classified by comparing their ribosomal D NA (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, Thirt y-nine rDNAs cloned after 35 cycles of PCR gave 13 sequences for 74% c overage, Thus, the representation of the ecosystem after 35 cycles of PCR was distorted arid lacked diversity, However, when the number of t emperature cycles was minimized, biodiversity was preserved, and there was good agreement between culturing bacteria and sampling rDNA direc tly.