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Applied and Environmental Microbiology, April 2008, p. 2461-2470, Vol. 74, No. 8
0099-2240/08/$08.00+0     doi:10.1128/AEM.02272-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Critical Evaluation of Two Primers Commonly Used for Amplification of Bacterial 16S rRNA Genes{triangledown}

Jeremy A. Frank,1,2 Claudia I. Reich,1,3 Shobha Sharma,2,{dagger} Jon S. Weisbaum,4,5 Brenda A. Wilson,1,2 and Gary J. Olsen1,2*

Department of Microbiology,1 Host-Microbe Systems Theme, Institute for Genomic Biology,2 National Center for Supercomputing Applications,3 College of Medicine, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801,4 Carle Foundation Hospital, Urbana, Illinois 618015

Received 5 October 2007/ Accepted 11 February 2008

rRNA-based studies, which have become the most common method for assessing microbial communities, rely upon faithful amplification of the corresponding genes from the original DNA sample. We report here an analysis and reevaluation of commonly used primers for amplifying the DNA between positions 27 and 1492 of bacterial 16S rRNA genes (numbered according to the Escherichia coli rRNA). We propose a formulation for a forward primer (27f) that includes three sequences not usually present. We compare our proposed formulation to two common alternatives by using linear amplification—providing an assessment that is independent of a reverse primer—and in combination with the 1492 reverse primer (1492r) under the PCR conditions appropriate for making community rRNA gene clone libraries. For analyses of DNA from human vaginal samples, our formulation was better at maintaining the original rRNA gene ratio of Lactobacillus spp. to Gardnerella spp., particularly under stringent amplification conditions. Because our 27f formulation remains relatively simple, having seven distinct primer sequences, there is minimal loss of overall amplification efficiency and specificity.

* Corresponding author. Mailing address: Department of Microbiology, University of Illinois at Urbana-Champaign, B103 C&LSL, 601 South Goodwin Ave., Urbana, IL 61801. Phone: (217) 244-0616. Fax: (217) 244-6697. E-mail: gary{at}life.uiuc.edu

{triangledown} Published ahead of print on 22 February 2008.

{dagger} Present address: Department of Physical and Environmental Sciences, University of Toronto at Scarborough, 1265 Military Trail, Toronto, ON, Canada M1C 1A4.

Applied and Environmental Microbiology, April 2008, p. 2461-2470, Vol. 74, No. 8
0099-2240/08/$08.00+0     doi:10.1128/AEM.02272-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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