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.
Steward
Grieg F
GF
Department of Oceanography, School of Ocean and Earth Science and Technology, University of Hawaii at Manoa, Honolulu, Hawaii 96822, USA.
Jenkins
Bethany D
BD
Ward
Bess B
BB
Zehr
Jonathan P
JP
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