Open AccessAdvantage of Using Inosine at the 3′ Termini of 16S rRNA Gene Universal Primers for the Study of Microbial Diversity
Author(s) -
Eitan BenDov,
Orr H. Shapiro,
Nachshon Siboni,
Ariel Kushmaro
Publication year - 2006
Publication title -
applied and environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.552
H-Index - 324
eISSN - 1070-6291
pISSN - 0099-2240
DOI - 10.1128/aem.00849-06
Subject(s) - biology , primer (cosmetics) , proteobacteria , 16s ribosomal rna , library , firmicutes , genetics , phylum , inosine , gene , ribosomal rna , computational biology , biochemistry , chemistry , enzyme , organic chemistry
To overcome the shortcomings of universal 16S rRNA gene primers 8F and 907R when studying the diversity of complex microbial communities, the 3′ termini of both primers were replaced with inosine. A comparison of the clone libraries derived using both primer sets showed seven bacterial phyla amplified by the altered primer set (8F-I/907R-I) whereas the original set amplified sequences belonging almost exclusively toProteobacteria (95.8%). Sequences belonging toFirmicutes (42.6%) andThermotogae (9.3%) were more abundant in a library obtained by using 8F-I/907R-I at a PCR annealing temperature of 54°C, whileProteobacteria sequences were more frequent (62.7%) in a library obtained at 50°C, somewhat resembling the result obtained using the original primer set. The increased diversity revealed by using primers 8F-I/907R-I confirms the usefulness of primers with inosine at the 3′ termini in studying the microbial diversity of environmental samples.