Development of a Universal Microarray Based on the Ligation Detection Reaction and 16S rRNA Gene Polymorphism To Target Diversity of Cyanobacteria

DNA samples.

The samples used to validate the probes included axenic strains kept in our culture collections, strains isolated from European lakes and a reservoir during this study (Table 1), and clones of environmental DNA libraries obtained from Lake Esch-sur-Sûre (Luxembourg) and Lake Tuusulanjärvi (Finland) (Table 2). The 16S rRNA gene of the cultured strains and clones was sequenced (unpublished data). In addition, the array was tested with an environmental DNA sample (Lake Tuusulanjärvi), which was isolated by the hot-phenol method (9). To verify the microarray results, the same environmental sample was analyzed by DGGE and cloning of the 16S rRNA gene.

Ligation probe design.

For the LDR, we designed specific probes for the 16S rRNA gene sequences of 19 different cyanobacterial groups. These groups were identified by using a cyanobacterial 16S rRNA gene alignment built with ARB software, version Beta 011107 (16). The alignment contained 281 sequences from public databases and 57 from this study in addition to the out-group Escherichia coli. All of these sequences were longer than 1,400 bp, except the two sequences of Antarctic Phormidium (about 1,350 bp) and 21 (of 42) sequences of Prochlorococcus marinus (about 1,250 bp). All sequences were aligned with CLUSTAL W (26) and ARB. The sequence alignment is available upon request. The phylogenetic analysis was performed with ARB by using the neighbor-joining (NJ) algorithm (22). From the sequence alignment, group-specific consensus sequences were obtained with a cutoff percentage of 75%. If a base at a given position occurred at a lower frequency than the cutoff percentage, it was replaced by an appropriate International Union of Pure and Applied Chemistry ambiguity code in the consensus sequence. The group-specific consensus sequences were imported to GCG Omiga, version 2.0 (Oxford Molecular Ltd.), for group-specific probe design. The probes were designed by following the LDR approach. After hybridization of a discriminating probe and a common probe to the target sequence, ligation occurs only if there is perfect complementarity between the two probes and the template, in this case, an amplified fragment of the 16S rRNA gene (Fig. 1). For this reason, the discriminating probes were designed to have 3′ ends unique to each of the 19 cyanobacterial groups. The common probes were located immediately after the discriminating probes according to the group-specific consensus sequences. An example of selection is shown in Fig. 2. To discard potentially unspecific probe pairs, we checked each probe pair (discriminating probe and common probe) by using the probe match tool of the ARB program. We also designed a probe pair (named UNICYANO) to detect the presence of any cyanobacteria in the sample. No significant self-annealing of the probe sequences was detected by computer analysis (data not shown). All probes were designed to have a theoretical melting temperature (T_m) between 63 and 68°C, calculated by using the Oligonucleotide Properties Calculator program (http://www.basic.nwu.edu/biotools/oligocalc.html).

We randomly selected 21 czip code sequences from those described by Gerry et al. (7) and Chen et al. (4). These czip codes were randomly assigned to the UNICYANO probe pair, the 19 group-specific probe pairs, and a positive control for the hybridization reaction. The latter was a Cy3-labeled czip code that has its own corresponding zip code in the universal array. As a negative control for the hybridization and LDR, double-distilled water was used instead of genomic DNA as the PCR template. The discriminating probes were labeled with Cy3 at the 5′ end. The common probes were phosphorylated at the 5′ end and carried the czip code at the 3′ end. When a probe sequence contained an ambiguity code, this base was replaced with inosine during oligonucleotide synthesis.

Universal array preparation.

The microarrays were prepared by using CodeLink slides (Amersham Biosciences, Piscataway, N.J.), designed to covalently immobilize amino (NH₂)-modified oligonucleotides. The 5′ NH₂-modified zip code oligonucleotides, carrying an additional poly(dA)₁₀ tail at their 5′ ends, were diluted to 25 μM in 100 mM phosphate buffer (pH 8.5). Spotting was performed by using a contact-dispensing system (MicroGrid II; BioRobotics, Huntingdon, United Kingdom). The printed slides were processed according to the manufacturer's protocols. Eight arrays per slide were generated. Quality control of the printed surfaces was performed by sampling one slide from each deposition batch. This slide was hybridized with 1 μM 5′ Cy3-labeled poly(dT)₁₀ in a solution containing 5× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.1 mg of salmon sperm DNA/ml at room temperature for 2 h and then washed for 15 min in 1× SSC. The fluorescent signal was controlled by laser scanning, as described below.

PCR amplifications from DNA samples.

The 16S rRNA gene and the internal transcribed spacer region were amplified with universal primer 16S27F (5′-AGAGTTTGATCMTGGCTCAG-3′) (6) and cyanobacterium-specific primer 23S30R (5′-CCTCGCCTCTGTGTGCCTAGGT-3′) (14, 25). The PCR amplifications were performed with a GeneAmp PCR system 9700 thermal cycler (Applied Biosystems, Foster City, Calif.). The reaction mixtures included 500 nM concentrations of each primer, 200 μM concentrations of each deoxynucleoside triphosphate, 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl₂, 50 mM KCl, 0.1% (wt/vol) Triton X-100, 1 U of DyNAzyme DNA polymerase II (Finnzymes, Espoo, Finland), and 5 to 8 ng of genomic DNA in a final volume of 50 μl. Prior to amplification, the DNA was denatured for 5 min at 95°C. Amplification consisted of 30 cycles at 94°C for 45 s, 57°C for 45 s, and 72°C for 2 min. After the cycles, an extension step (10 min at 72°C) was performed. The PCR products were purified by using a GFX PCR DNA purification kit (Amersham), eluted in 50 μl of autoclaved water, and quantified with a BioAnalyzer 2100 (Agilent Technologies, Palo Alto, Calif.).

LDR.

The LDR was carried out in a final volume of 20 μl containing 20 mM Tris-HCl (pH 7.5), 20 mM KCl, 10 mM MgCl₂, 0.1% NP-40, 0.01 mM ATP, 1 mM dithiothreitol, 250 fmol of each discriminating probe, 250 fmol of each common probe, 10 fmol of the hybridization control, and from 0.5 to 100 fmol of purified PCR products. After the reaction mixture was preheated for 2 min at 94°C and centrifuged for 1 min, 4 U of Pfu DNA ligase (Stratagene, La Jolla, Calif.) was added. The LDR was cycled for 30 rounds at 90°C for 30 s and at 60°C for 4 min in a GeneAmp PCR system 9700 thermal cycler.

Array hybridization, detection, and data analysis.

The hybridization mixture had a total volume of 65 μl and contained 20 μl of LDR mixture, 5× SSC, and 0.1 mg of salmon sperm DNA/ml. After heating at 94°C for 2 min and chilling on ice, the hybridization mixture was applied to the slide, on which the eight arrays were separated by Press-To-Seal silicone isolators (1.0 × 9 mm; Schleicher & Schuell BioScience, Dassel, Germany). Hybridization was carried out in a chamber in the dark at 65°C for 1 h in a temperature-controlled water bath. After hybridization, the slide was washed at 65°C for 15 min in prewarmed 1× SSC and 0.1% sodium dodecyl sulfate. Finally, the slide was dried by spinning at 80 × g for 3 min. The fluorescent signals were acquired at a 5-μm resolution by using a ScanArray 4000 laser-scanning system (PerkinElmer Life and Analytical Sciences, Boston, Mass.) with a green laser for Cy3 dye (λ_ex, 543 nm; λ_em, 570 nm). Both the laser and the photomultiplier (PMT) tube power were set between 70 and 95%, depending on the signal intensities. QuantArray quantitative microarray analysis software (PerkinElmer) was used to quantitate the fluorescent intensity of the spots. The fluorescent intensity values obtained from the replicated spots (four replicate spots for each group-specific probe and eight replicates for the universal probe) and replicated experiment sets (three separate LDR-universal array experiments) were analyzed, and the means and standard deviations were calculated.

Concerning the method used to calculate nonspecific hybridization values, data analysis for each target was performed as follows: (i) the hybridization fluorescent intensities from nonspecific zip codes were calculated and averaged; (ii) the mean of these nonspecific hybridization values was compared with that of the expected positive zip code.

Sequence analysis of cyanobacterial 16S rRNA genes and design of ligation probes.

The cyanobacterial groups identified with ARB by using the NJ alghorithm (as described in Materials and Methods) were named after the genus designations of their components: Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, P. marinus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia (Fig. 3). The list of strains present in each group is available at http://www.ulg.ac.be/cingprot/midichip/output/publications/Castiglioni_Tree.htm. For all of these, a group-specific consensus sequence was determined and used for probe design. The probes were designed to be complementary to the polymorphic regions of the group-specific consensus sequence alignment. We selected 19 group-specific probe pairs and a universal control probe matching all of the cyanobacteria. All of the probes had theoretical melting temperatures between 63 and 68°C. Table 3 lists all of the selected group-specific and universal probes and randomly chosen czip code sequences. Although DNA samples for some of the 19 selected groups (Gloeothece, Antarctic Phormidium, Trichodesmium, and Cylindrospermopsis) were not available, they were included to allow future applications of this cyanobacterial microarray.

Validation of universal array designed for cyanobacteria. (i) Specificity of probes.

In the presence of a proper DNA template, only group-specific spots, universal spots, and those spots corresponding to the hybridization control showed positive signals. Several examples of the results are shown in Fig. 4. The specificity of the probes for freshwater cyanobacterial groups was tested by using PCR-amplified 16S rRNA genes originating either from 52 cyanobacterial strains (both axenic and isolated in this study) or from 44 clones. Three replicated LDR-universal array experiments showed good reproducibility of the results.

The intensities of signals of nonspecific hybridization for the cyanobacterial groups examined never exceeded 6% with respect to the expected positive signals (Table 4), and this value was used as the lower limit for positive signals in subsequent microarray analyses.

To evaluate the relative ligation efficiency of the probes, the mean signal intensity values of the group-specific spots for each target were measured and normalized with respect to the signal intensity values of the universal spot. The hybridization intensities of the probes differed and ranged from 92 to 155% (Table 4).

A negative control of the entire process was performed by using double-distilled water instead of genomic DNA as the PCR template. Following hybridization on the universal chip, no signal was detected even after setting the PMT and laser to 95% power (data not shown).

(ii) LDR sensitivity.

To establish the detection limit of the method and the correlation between signal intensity and template concentration, we tested various template concentrations (0.5 to 100 fmol) in the LDR. The PCR products originated from the strains Planktothrix sp. strain 1LT27S08, Calothrix sp. strain PCC 7714, and Microcystis aeruginosa PCC 9354. The detected signals progressively decreased, and signal was detectable in up to 1 fmol of the PCR product, corresponding to 1 ng of amplified DNA. No signals were detected with 0.5 fmol of the PCR product, even after setting the PMT and laser to 95% power (data not shown). We found a linear correlation (R² = 0.98, 0.94, and 0.96, respectively) between signal intensity and template concentration (Fig. 5A). Nevertheless, the signal-to-noise ratio also decreased with gradually reducing template concentration (Fig. 5B). This ratio was obtained from the signal intensities of the target-specific spots divided by the mean signal intensity of the nonspecific spots. The signal-to-noise ratio was clearly higher at template quantities above 25 fmol than at lower quantities (Fig. 5B). Essentially the same results were obtained with similar concentrations of PCR products derived from Calothrix sp. strain PCC 7714 and M. aeruginosa PCC 9354 as the LDR substrate (data not shown). Therefore, the target concentration of 25 fmol of each strain or clone was used in the LDR.

Use of artificial mixes of PCR products from different strains.

To determine the efficiency of the LDR method in the presence of complex molecular targets, we used artificial mixes with unequal amounts of PCR products derived from the following cyanobacterial strains: M. aeruginosa PCC 9354, Aphanizomenon sp. strain 202, Planktothrix sp. strain 1LT27S08, Spirulina major sp. strain PCC 6313, and Calothrix sp. strain PCC 7714. After separate PCRs, the amplified fragments were pooled in the unbalanced mixes. In all of these experiments, the unbalanced mixes were 5 fmol of both S. major and Calothrix versus 100 fmol of both Aphanizomenon and Microcystis or Planktothrix. After hybridization of the LDR products on the universal array, all of the expected signals were detected and easily discriminated from the nonspecific signals. An example of these experiments is shown in Fig. 6A. The amplicon concentrations were reflected in the signal intensities (Fig. 6A).

LDR detection on universal array of the 16S rRNA gene from an environmental sample.

The 16S rRNA gene from a sample collected from Lake Tuusulanjärvi was analyzed to evaluate the DNA microarray applicability for environmental studies. Microarray hybridization patterns showed the presence of Microcystis, Anabaena/Aphanizomenon, and Woronichinia spp. (Fig. 6B). In DGGE, the following cyanobacterial groups were detected: Microcystis, Snowella, and Anabaena/Aphanizomenon. Cloning revealed the following groups: Microcystis (the most abundant group, 62% of the cyanobacterial clones), Anabaena/Aphanizomenon (18%), and Snowella (15%) (A. Rantala, P. Rajaniemi, and K. Sivonen, unpublished data).