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Miniprimer PCR, a New Lens for Viewing the Microbial World ^,

Thomas A. Isenbarger,^1,2 Michael Finney,^2, Carlos Ríos-Velázquez,³ Jo Handelsman,^1* and Gary Ruvkun²

Departments of Bacteriology and Plant Pathology, University of Wisconsin—Madison, 1550 Linden Drive, Madison, Wisconsin 53706-1521,¹ Department of Genetics, Harvard University, and Department of Molecular Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114-2790,² Department of Biology, University of Puerto Rico—Mayagüez, Mayagüez, Puerto Rico³

Received 22 August 2007/ Accepted 29 November 2007

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.

Published ahead of print on 14 December 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

Present address: Finney Capital, 489 Douglass St., San Francisco, CA 94114.