Miniprimer PCR, a new lens for viewing the microbial world

Departments of Bacteriology and Plant Pathology, University of Wisconsin-Madison, 2Department of Genetics, Harvard University, and Department of Molecular Biology, Massachusetts General Hospital Department of Biology, University of Puerto Rico-Mayagüez

^* To whom correspondence should be addressed. Email: joh{at}bact.wisc.edu.

	Abstract

Molecular methods based on the 16S ribosomal RNA 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-nt "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 ribosomal RNA 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.