Pushing the limits for amplifying BrdU-labeled DNA encoding 16S rRNA: DNA polymerase as the determining factor.
Identifying microorganisms that are active under specific conditions in ecosystems is a challenge in microbial ecology. Recently, the bromodeoxyuridine (BrdU) technique was developed to label actively growing cells. BrdU, a thymidine analog, is incorporated into newly synthesized DNA, and the BrdU-labeled DNA is then isolated from total extractable DNA by immunocapture using a BrdU-specific antibody. Analyzing the BrdU-labeled DNA allows for assessing the actively growing community, which can then be compared to the unlabeled DNA that represents the total community. However, applying the BrdU approach to study soils has been problematic due to low DNA amounts and soil contaminants. To address these challenges, we developed a protocol, optimizing specificity and reproducibility, to amplify BrdU-labeled gene fragments encoding 16S rRNA. We found that the determining factor was the DNA polymerase: among the 13 different polymerases we tested, only 3 provided adequate yields with minimal contamination, and only two of those three produced similar amplification patterns of community DNA.
Roux-Michollet
Dad D
DD
Institute for Computational Earth System Science, University of California, Santa Barbara, CA 93106, USA. droux@bren.ucsb.edu
Schimel
Joshua P
JP
Holden
Patricia A
PA
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001973:Bromodeoxyuridine / Q000032:analysis / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000737:chemistry / Q000235:genetics*; D012987:Soil / Q000032:analysis; D012988:Soil Microbiology; D013194:Staining and Labeling / Q000295:instrumentation / Q000379:methods*
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012987:Soil; D004259:DNA-Directed DNA Polymerase; D001973:Bromodeoxyuridine