Evaluation of primers and PCR conditions for the analysis of 16S rRNA genes from a natural environment.
We investigated biases occurring in the polymerase chain reaction (PCR) amplification of 16S rRNA genes from an environmental sample, by comparing the clone libraries that we had previously prepared from the gut homogenate of the termite Reticulitermes speratus. We detected a significant increase in the expected number of phylotypes by lowering the annealing temperature, and a significant decrease in the proportion of clones belonging to the predominant group by raising the number of PCR cycles. We also found that the Bacteria-universal primer, 63F, introduced a seriously biased amplification caused by primer mismatches, in contrast to a previous report. These results, together with suggestions from previous studies using simplified model samples, will help us to recognize the limitations of PCR-based analysis.
Hongoh
Yuichi
Y
International Cooperative Research Project, Japan Science and Technology Corporation (JST-ICORP), Saitama, Japan. yhongo@postman.riken.go.jp
Yuzawa
Hiroe
H
Ohkuma
Moriya
M
Kudo
Toshiaki
T
D000818:Animals; D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000032:analysis; D004783:Environmental Microbiology; D015723:Gene Library; D020049:Isoptera / Q000382:microbiology*; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012336:RNA, Ribosomal, 16S / Q000032:analysis* / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D013696:Temperature
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S