In:
Applied and Environmental Microbiology, American Society for Microbiology, Vol. 70, No. 3 ( 2004-03), p. 1455-1465
Abstract:
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.
Type of Medium:
Online Resource
ISSN:
0099-2240
,
1098-5336
DOI:
10.1128/AEM.70.3.1455-1465.2004
Language:
English
Publisher:
American Society for Microbiology
Publication Date:
2004
detail.hit.zdb_id:
223011-2
detail.hit.zdb_id:
1478346-0
SSG:
12
