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Applied and Environmental Microbiology, March 2004, p. 1455-1465, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1455-1465.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Development and Testing of a DNA Macroarray To Assess Nitrogenase (nifH) Gene Diversity

Grieg F. Steward,1 Bethany D. Jenkins,2 Bess B. Ward,3 and Jonathan P. Zehr2*

Department of Oceanography, School of Ocean and Earth Science and Technology, University of Hawaii at Manoa, Honolulu, Hawaii 96822,1 Ocean Sciences Department and Institute of Marine Sciences, University of California, Santa Cruz, Santa Cruz, California 95064,2 Department of Ecology and Evolutionary Biology, Princeton University, Princeton, New Jersey 085443

Received 26 August 2003/ Accepted 1 December 2003

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml-1, a half-saturation of signal at 0.26 ng ml-1, and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.

* Corresponding author. Mailing address: Ocean Sciences Department and Institute of Marine Sciences, University of California, Santa Cruz, Santa Cruz, CA 95064. Phone: (831) 459-4009. Fax: (831) 459-4882. E-mail: zehrj{at}cats.ucsc.edu.

Applied and Environmental Microbiology, March 2004, p. 1455-1465, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1455-1465.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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