Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity.
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.
Department of Oceanography, School of Ocean and Earth Science and Technology, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA.
Grieg F
GF
Steward
Bethany D
BD
Jenkins
Bess B
BB
Ward
Jonathan P
JP
Zehr
D001105:Archaea / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D001483:Base Sequence; D019641:DNA, Archaeal / Q000235:genetics / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial; D014644:Genetic Variation; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation / Q000235:genetics*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010088:Oxidoreductases / Q000235:genetics*; D014871:Water Microbiology
D019641:DNA, Archaeal; D004269:DNA, Bacterial; D010088:Oxidoreductases; C029130:nitrogenase reductase