e166 Nucleic Acids Research, 2005, Vol. 33, No. 19 endophytic bacterium Azoarcus sp. strain BH72 (AF200742) (7). When stated otherwise, nifH of Azoarcus indigens VB32T (8) or vnfH of Azotobacter vinelandii DSMZ 366 was used. End-labelled targets were obtained by PCR using three differ- ent primer pairs, generating three amplicons with different lengths (Z1 ¼ 362 nt length, position 737–1098 nt on AF200742; Z2 ¼ 322 nt length, position 737–1058; Z3 ¼ 209 nt length, position 850–1058 nt). Chromosomal bacterial DNA used as a template was puriﬁed by standard procedures (9). Degenerate oligonucleotides typically used for ampliﬁcation of divergent nitrogenase genes (nifH, anfH and vnfH ) (10) were applied to amplify target Z1, the forward primer being labelled with Cy5, Cy3 or biotin, respectively, at the 50 end [Z1-nifH-F: 50-TG(CT) GA(CT) CC(AGCT) AA(AG) GC(AGCT) GA-30], and the reverse primer being modiﬁed with biotin or Cy3, respectively, at the 50 end [Z1-nifH-R: 50-A(AGT)(ACGT) GCC ATC AT(CT) TC(ACGT) CC-30]. For the shortened fragments Z2 and Z3, primers were designed to speciﬁcally amplify nifH of Azoar- cus sp. strain BH72 [Z2-nifH-F-Cy5, 50-TGC GAC CCG AAG GCT GA-30, and Z2-nifH-R-Biotin, 50-GGG CCT TGT TTT CGC G-30; Z3-nifH-F-Cy5, 50-CCT GTC GGT CGG CTT C- 30, and Z2-nifH-R-Biotin, 50-GGG CCT TGT TTT CGC G-30]. Ampliﬁcation reactions were performed as described previ- ously (11). Ampliﬁcation of a nifD fragment (365 nt length, position 2109–2473 nt on AF200742) (7) was carried out with primer nifD-BH72-F labelled with Cy5 at the 50 end [ATC GGC GAC GAC ATC GAA GCC] and biotinylated primer nifD-BH72-R [TAG TTC ATC GAG CGG TAG CAG T]. Ampliﬁcation conditions were an initial denaturing for 5 min at 95 C, then 40 cycles of 95 C for 1 min, 60 C for 1 min, 72 C for 1 min, with a ﬁnal extension at 72 C for 5 min. Successful ampliﬁcation was conﬁrmed by analysing PCR products on 2% agarose gels, and fragments were puriﬁed with the QIAquick PCR puriﬁcation kit (Qiagen, Hilden, Germany). For hybridization, ﬂuorescence end-labelled single- stranded DNAs (ssDNAs) were isolated from the double- stranded DNA ampliﬁcation products. Strands were separated with streptavidin-coated paramagnetic particles (Roche, Mannheim, Germany) as described previously (12), and the concentration of ssDNA was determined by UV spectrometry. Shortly before use, the ssDNA was routinely denatured at 95 C for 10 min and then snap-cooled on ice. Alternatively, the nifH target was end-labelled with [g-33P]ATP (25 TBq/mmol; Hartmann Analytics, Braunschweig, Germany) by using T4 polynucleotide kinase (Fermentas, St Leon-Roth, Germany) according to manufac- turer’s instructions; the probe was separated from unincorpor- ated label by gelﬁltration with P6 Biogel. Random-labelled target Unlabelled Z2 sense was generated by PCR using unlabelled primer Z2-nifH-F and primer Z2-nifH-R-Biotin. After strand separation, ssDNA was used for multiple random ﬂuorescent labelling with Alexa Fluor 647 included in ULYSIS nucleic acid labelling kit (Molecular probes, Inc., Eugene, Oregon) following the manufacturer’s instructions. Shortly before use, the ﬂuorescent-labelled ssDNA was routinely denatured at 95 C for 10 min and then snap-cooled on ice. PAGE 2 OF 8 Oligonucleotide probes All oligonucleotides targeting the nifH gene of Azoarcus sp. strain BH72 were designed with GeneDoc [K. B. Nicholas and H. B. J. Nicholas (1997) http://www.psc.edu/biomed/ genedoc] according to the following criteria: their binding region had similar characteristics with respect to length (16–18 nt), %GC content, melting temperature (Tm) calculated with MELT 1.1.0 (J. P. Sanders), and DG taking into account the complementary strand. Their sequence is shown in Table 1 and their relative position with respect to the target fragment in Figure 1. They all carried polyadenosine triphosphate spacers (6 A), had either a 50- or 30-amino modiﬁcation (Aminolink C6), and were synthesized by Thermo Electron GmbH (Ulm, Germany). Secondary structure predictions for the target strands were carried out using the Mfold program (13). The same criteria and methods were used to design oligonuc- leotides targeting a different gene, nifD of strain BH72, des- ignated as nifD-A1 and so forth (Table 1). As examples of longer oligonucleotide probes, 50mers speciﬁc for the nifH gene of Azoarcus sp. strain BH72 (7) were designed: A1– 50 (position 737–786 nt on AF200742), A19–68 (position 755–804 nt on AF200742) and A174–223 (position 910– 959 nt on AF200742). Fabrication of microarrays The DNA microarray format used in this study was based on standard microscopic glass slides (Menzel, Braunschweig, Germany). Chemicals and solvents were from Fluka (Neu- Ulm, Germany). The substrates were cleaned, silylated and activated (isothiocyanate-functionalized surface) as described by Beier and Hoheisel (14). The activated surfaces were used directly for immobilization of either 50- or 30-amino-modiﬁed capture oligonucleotides via covalent binding. Probes were spotted onto the activated slide surface using the piezo- driven spotting device Robodrop (BIAS, Bremen, Germany) bearing a glass pin, or MicroGrid II Compact 400 (Genomic Solutions Ltd, Huntingdon, Cambridgeshire) with an ArrayIt split pin (TeleChem International, Inc., Sunnyvale, CA), respectively. The concentration of the amino-modiﬁed oligonucleotides in 1% glycerol was 10 mM. Volumes of deposited oligonucleotide solutions were 200 pl, resulting in spots with a diameter of 200 mm. To complete covalent binding, after being spotted with probe solutions, slides were incubated overnight at room temperature in a wet chamber to prevent evaporation of the spots. Blocking of the slides was performed in 6-amino-1-hexanol (50 mM) and diisopropy- lethylamine (150 mM) in dimethylformamide (15). Five types of commercial slides with different chemically modiﬁed surfaces were used for comparison. Three types of slides were purchased from Genetix (Dornach, Germany) with aldehyde, amine or ‘aldehyde plus’ surface; one was pur- chased from PEQLAB Biotechnologie (Erlangen, Germany) with amino surface coating (QMT Amino Slides); one amine slide (Pan Amine slide) was purchased from MWG Biotech (Ebersberg, Germany). Spotting and hybridization of microar- rays were performed following manufacturer’s instructions. Microarray hybridization and evaluation Hybridization and washing were done in a PersonalHyb oven (Stratagene). Hybridization time varied from 1 h to overnight,