Miniprimer PCR, a new lens for viewing the microbial world.
Molecular methods based on the 16S rRNA gene sequence are used widely in microbial ecology to reveal the diversity of microbial populations in environmental samples. Here we show that a new PCR method using an engineered polymerase and 10-nucleotide "miniprimers" expands the scope of detectable sequences beyond those detected by standard methods using longer primers and Taq polymerase. After testing the method in silico to identify divergent ribosomal genes in previously cloned environmental sequences, we applied the method to soil and microbial mat samples, which revealed novel 16S rRNA gene sequences that would not have been detected with standard primers. Deeply divergent sequences were discovered with high frequency and included representatives that define two new division-level taxa, designated CR1 and CR2, suggesting that miniprimer PCR may reveal new dimensions of microbial diversity.
Isenbarger
Thomas A
TA
Department of Bacteriology, University of Wisconsin-Madison, 1550 Linden Drive, Madison, WI 53706-1521, USA.
Finney
Michael
M
Ríos-Velázquez
Carlos
C
Handelsman
Jo
J
Ruvkun
Gary
G
D019295:Computational Biology; D017931:DNA Primers / Q000235:genetics*; D019641:DNA, Archaeal / Q000032:analysis / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D019015:Geologic Sediments / Q000382:microbiology*; D006217:Halobacterium / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology; D019914:Taq Polymerase / Q000235:genetics
D017931:DNA Primers; D019641:DNA, Archaeal; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase