Open AccessComplementary retrieval of 16S rRNA gene sequences using broad‐range primers with inosine at the 3′‐terminus: implications for the study of microbial diversity
Author(s) -
Brands Berit,
Vianna Morgana E.,
Seyfarth Ilse,
Conrads Georg,
Horz HansPeter
Publication year - 2010
Publication title -
fems microbiology ecology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.377
H-Index - 155
eISSN - 1574-6941
pISSN - 0168-6496
DOI - 10.1111/j.1574-6941.2009.00786.x
Subject(s) - biology , inosine , primer (cosmetics) , terminal restriction fragment length polymorphism , 16s ribosomal rna , genetics , gene , ribosomal rna , amplicon , restriction fragment length polymorphism , microbiology and biotechnology , computational biology , polymerase chain reaction , biochemistry , chemistry , organic chemistry , enzyme
We evaluated the impact of the base analogue inosine substituted at the 3′‐terminus of broad‐range 16S rRNA gene primers on the recovery of microbial diversity using terminal restriction fragment length polymorphism and clonal analysis. Oral plaque biofilms from 10 individuals were tested with modified and unmodified primer pairs. Besides a core overlap of shared terminal restriction fragments (T‐RFs), each primer system provided unique information on the occurrence of T‐RFs, with a higher number generally displayed with inosine primers. All clones sequenced were at least 99% identical to publicly available full‐length sequences. Analysis of the corresponding primer‐binding sites showed that most sequence types were 100% complementary to the unmodified primers so that the characteristic of inosine to bind with all four nucleotides was not crucial for the observed increase in microbial richness. Instead, differences in community compositions were correlated with the identity of the nearest‐neighbor 3′ of the primer‐targeting region. By influencing the thermal stability of primer hybridization, this position may play a previously unrecognized role in biased amplification of 16S rRNA gene sequences. In conclusion, the combined use of inosine and unmodified primers enables the complementary retrieval of 16S rRNA gene types, thereby expanding the observed diversity of complex microbial communities.