A nested PCR approach for improved recovery of archaeal 16S rRNA gene fragments from freshwater samples.
In a survey on the presence of archaea in a number of European lakes, it was found that known archaeal primer sets for PCR were not suited for use in freshwater environment, as some lack selectivity, while others were too selective. A nested PCR was developed for denaturing gradient gel electrophoresis (DGGE) with primer sets 21F-958R and Parch519f-Arch915r, respectively. After sequencing of the DGGE bands obtained by this nested method, 93% of the sequences were of archaeal origin. More diverse archaeal DGGE patterns were found as compared with other PCR methods. The nested PCR-DGGE method presented here is therefore a reliable tool to analyze the archaeal diversity in freshwater habitats, revealing even more widespread diversity of the archaea.
Vissers
Elisabeth W
EW
Department of Microbial Wetland Ecology, Centre for Limnology, Netherlands Institute of Ecology (NIOO-KNAW), Nieuwersluis, The Netherlands. l.vissers@nioo.knaw.nl
Bodelier
Paul L E
PL
Muyzer
Gerard
G
Laanbroek
Hendrikus J
HJ
D001105:Archaea / Q000235:genetics / Q000302:isolation & purification*; D016000:Cluster Analysis; D017931:DNA Primers / Q000235:genetics; D019641:DNA, Archaeal / Q000235:genetics / Q000302:isolation & purification*; D004275:DNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005618:Fresh Water / Q000382:microbiology*; D008969:Molecular Sequence Data; D009691:Nucleic Acid Denaturation; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D019641:DNA, Archaeal; D004275:DNA, Ribosomal