PAGE 3 OF 8 Table 1. Oligonucleotide probes used in this study Probesa A1 A4 A8 A14 A20 A64 A114 A188 A235 A307 S20 S64 S114 S188 S235 S307 A20-30 A307-30 nifD-A1 nifD-A8 nifD-A77 nifD-A185 nifD-A307 VB-A34 VB-A164 VB-A307 vnf-A47 vnf-A110 vnf-A309 Sequences (50–30)b AAAAAA TCAGCCTTCGGGTCGCA AAAAAA GAGTCAGCCTTCGGGTC AAAAAA GGTGGAGTCAGCCTTCG AAAAAA CAGACGGGTGGAGTCAG AAAAAA GAGGATCAGACGGGTGG AAAAAA CTTCAGCGGCCAGGTG AAAAAA GAAGCCGACCGACAGG AAAAAA GGCGGTGATAACGCCAC AAAAAA GAAGTCGAGTTCGTCGTC AAAAAA GGGCCTTGTTTTCGCG AAAAAA CCACCCGTCTGATCCTC AAAAAA CACCTGGCCGCTGAAG AAAAAA CCTGTCGGTCGGCTTC AAAAAA GTGGCGTTATCACCGCC AAAAAA GACGACGAACTCGACTTC AAAAAA CGCGAAAACAAGGCCC GAGGATCAGACGGGTGG AAAAAA GGGCCTTGTTTTCGCG AAAAAA AAAAAA TTCGATGTCGTCGCCGAT AAAAAA AGACGGCTTCGATGTCGT AAAAAA ACACGCCACGGAAGCCTT AAAAAA CGATGATGGTGACGTCGT AAAAAA CTTCGGGGTGTTCTCCAT AAAAAA TGTGCCTTGCTGTGCAG AAAAAA GCAGCCGACGCCGGGTT AAAAAA GGGCCTTGTTTTCGCG AAAAAA ATGACGGTGCCCTGGG AAAAAA GCCGATCTGCAGCACGT AAAAAA TCCTGGGCCTTGTTCTCG Nucleic Acids Research, 2005, Vol. 33, No. 19 e166 Tm ( C)c Length (nt) GC% 68 68 68 68 68 68 68 68 67 65 68 68 68 68 67 65 68 65 67 67 69 67 67 66 71 65 68 68 69 17 17 17 17 17 16 16 17 18 16 17 16 16 17 18 16 17 16 18 18 18 18 18 17 17 16 16 17 18 64 64 64 64 64 68 68 64 55 62 64 68 68 64 55 62 64 62 55 55 61 55 55 58 76 62 68 64 61 Positiond 1–17 4–20 8–24 14–30 20–36 64–79 114–129 188–204 235–252 307–322 327–343 284–299 234–249 160–176 113–130 41–56 20–36 307–322 1–18 8–25 77–94 185–202 307–324 34–50 164–180 307–322 47–62 110–126 309–326 aA and S represent antisense reverse or sense probes, respectively, the numbers indicating the corresponding regions in the target fragment of strain BH72: e.g. S20 corresponds to sequence 20–36 nt of the sense strand, and A20 is reverse complementary to S20; 50 or 30: the oligonucleotide was amino-modified on the 50 or 30 end, respectively. bPolyadenosine triphosphate spacer (6A) on either 50 or 30 end shown with gap. c dTPmosvitaiolunessowfethree pcraolcbuelsawteedrewsithhowMnEaLcTco1r.d1i.n0g(tJootPh.eSbainnddienrgs)reagtiaonsaslotncothnececnotmraptiloenmoefnt6a0ry0 tmarMge.ts. Binding positions of sense probes on the antisense target were shown in italics. Figure 1. Schematic representation of the probe positions relative to the targets. Azoarcus sp. BH72 nifH Z-fragments of different lengths amplified from genomic DNA of Azoarcus sp. BH72 were used as fluorescence-labelled targets. The regions harbouring primers for amplification by PCR are shown as dotted lines. Short solid lines represent the regions where the probes were selected; e.g. S20 was designed from the region between 20 and 36 nt of the sense strand, and A20 is reverse complementary to S20. The fluorescent label is always located at the 50 end of the strand and shown as a star. without signiﬁcantly different results. Incubation was performed at 25 C. The ﬁnal concentration of target ssDNA in hybridization buffer [4· SET (9), 10· Denhardt’s solution and 50% formamide) was 10 nM. To guarantee a uniform moistening of the slide surface, the sample was covered with a cover slip. Washing was done routinely with 2· SET/0.1% SDS for 5 min and 1 · SET/0.1% SDS for 10 min at room temperature. Spin-dried slides were imaged at a resolution of 10 mm with a GenePix4000A microarray scanner (Axon, Union City, CA) at the same laser power and