Retrieval of nearly complete 16S rRNA gene sequences from environmental DNA following 16S rRNA-based community fingerprinting.
16S rRNA-based fingerprinting techniques allow rapid analyses of overall bacterial community structure but suffer from a lack of phylogenetic information hitherto retrievable from the short 16S rRNA gene sequences obtained from excised bands. An approach is presented that allows nearly complete 16S rRNA gene sequences to be retrieved for abundant components of the bacterial community as obtained by the community fingerprint, i.e. those reflected by major fingerprint bands. This was achieved by designing a pair of highly specific primers derived from the sequence of an excised band. Combined with universal 16S rRNA primers, these specific primers were applied directly to environmental DNA serving as template. This procedure allowed the generation of a nearly complete 16S rRNA gene sequence of the target taxon by specific polymerase chain reaction (PCR) followed by cycle sequencing down to a relative abundance of at least 1.5% of the environmental DNA. The procedure was exemplified for an epsilonproteobacterium related to Thiomicrospira denitrificans occurring in the central Baltic Sea. This approach is based only on PCR without any cloning step involved. It allows focussing on specific target taxa and is thus rather efficient. This approach should be applicable in general to 16S rRNA or 16S rRNA gene-based fingerprinting techniques and their respective environmental DNA.
Höfle
Manfred G
MG
GBF-German Research Centre for Biotechnology, Department of Environmental Microbiology, Mascheroder Weg 1, D-38124 Braunschweig, Germany.
Flavier
Sébastien
S
Christen
Richard
R
Bötel
Julia
J
Labrenz
Matthias
M
Brettar
Ingrid
I
D016172:DNA Fingerprinting / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics; D020565:Epsilonproteobacteria / Q000235:genetics*; D009792:Oceans and Seas; D018807:Polymorphism, Single-Stranded Conformational; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014871:Water Microbiology
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S