DNA chips for phylogenetic probing
method development
Katja Kerkmann§ and Linda Medlin*§
§Alfred
Wegener Institute for Polar- and Marine Research, Am Handelshafen 12, D-27570
Bremerhaven
Abstract
Marine picoeukaryotes provide an important contribution to the biomass production in
open oceanic regions. However due to technical limitations of conventional methods
e.g. electron microscopy there is hardly knowledge about the composition,
distribution and abundance of picoplankton. These limitations can be overcome by
using molecular techniques to study the picoplankton composition. DNA microarray
technology has the potential to quickly analyse the complex picoplankton samples.
We present the results of a DNA-chip dedicated to the identification of picoplankton
at the level of division, class or clade. The probes used on the DNA-chip were initially
designed for other hybridisation techniques, such as dot-blot hybridisation or FISH
(fluorescent in situ hybridisation). It appears that after a hybridisation of the
corresponding target DNA not all of the chosen probes result in a significant signal.
We show that there is a positional effect on the signal intensities if the 18S-DNA is
hybridised to probes immobilised on a DNA-chip.
Introduction
It is widely accepted that the phytoplankton community in the open ocean is
dominated by cells with a size smaller than 2-3 µm, the picoplankton (Li et al. 1994).
Especially
the
eukaryotes
among
the
picoplanktonic
organisms
contribute
significantly to both biomass and production in open oceanic regions (Campbell et al.,
1994; Li 1994), they are probably the most abundant eukaryotes on earth.
Nevertheless the taxonomic composition of marine picoeukaryotes is not very well
understood and there is hardly knowledge about the distribution and abundance of
the different taxa in situ (Partensky et al., 1997).
It seems that marine
picoeukaryotes belong to a wide range of different phylogenetic groups.
Picoeukaryotes are found in almost every algal phylum (Thomsen 1986) and new
algal classes have been described in the past decade (Andersen et al., 1993; Guillou
et al., 1999). With conventional methods e.g. electron microscopy it is possible to
discriminate the picoplankton at the class level, but a lack of ultrastructural features
makes the identification at lower taxonomic levels very difficult (Partensky et al.,
1997). In the past years the analysis of small-subunit (SSU) rRNA genes has been
established as an alternative way to analyse eukaryotic picoplankton- diversity. A
number of publications addressing questions considering the biodiversity of
picolankton with this approach has been launched in the past decade ( Díez et al.,
2001; Moon-van der Staay et al., 2001). Direct cloning and sequencing of the small
subunit (SSU) ribosomal DNA (rDNA) from natural samples makes it possible to get a
whole view of the structure and composition of the picoplankton community (LópezGarcia et al., 2001). This method circumvents the selective step of laboratory
cultivation (Giovannoni et al., 1990). The continually growing number of available
algal 18S-rDNA-sequences e.g. in the Ribosomal Database Project (RDP) (Maidak et
al., 2001) makes it possible to design probes that specifically target the 18S-rDNA or
rRNA (Lange et al., 1996; Guillou et al., 1999) down to species level. The probes
specific for the 18S-sequence are applied for the analysis of phytoplankton
communities by methods that take advantage of the hybridisation-principle e.g. flow
cytometry or epifluorescence microscopy (Miller and Scholin 1998; Lim et al., 1999).
Since these well established approaches are only suited to identify one or a few
organisms at a time (De Long et al., 1989), they are from limited use if larger
numbers of species have to be identified. Which makes it very time-consuming to get
a whole view of a picoplankton sample. This limitation can be overcome by using the
recently developed DNA-microarray-technology, which allows the simultaneous
analysis of thousands of probes at a time (Lockhart et al., 1996). At the heart of the
DNA-microarray technology is a DNA-microchip that contains an array of
oligonucleotides, PCR-products or cDNAs spotted onto a small surface. Target
nucleic acids are labeled with a fluorescent dye prior to their hybridisation to the
DNA-chip. The fluorescence pattern on the DNA-chip after the hybridisation of the
target-DNA to the probes spotted onto the microchip is then analysed with a
fluorescent laser-scanner (DeRisi et al., 1997). DNA-microarray technology has
enormous potential to be used as a method to analyse samples from complex
environments, because it provides a tool to analyse samples quickly without a
cultivation step. The first DNA-microchip to study microbial diversity was a chip to
analyse samples that contained nitrifying bacteria, which are difficult to study by
cultivation (Guschin et al., 1997). In that publication a hierarchical set of
oligonucleotide probes targeting the 16S ribosomal rRNA (rRNA) was created to
analyse the bacterial samples on the DNA-chip. Here we present data from our EUproject PICODIV that aims to use microarray technology for the analysis of
phytoplankton samples, by transferring probes that originally have been developed
and used for FISH onto micorarrays.
Material and Methods
Algal strains and Templates. Genomic DNA or plasmid-DNA was used as a
template for the amplification of the 18S-DNA. The plasmid-DNA consisted of a
~1800 bp 18S- PCR-fragment cloned into a plasmid-vector. The clones were isolated
in the EU FP5- Project PICODIV. The plasmid-DNA was isolated from bacteria by
using the Qiagen Plasmid Mini Kit (Qiagen, Germany). Genomic DNA was extracted
from pure cultures with the DNeasy Plant Mini Kit (Qiagen, Germany).
PCR amplification of 18S rRNA. For subsequent microarray hybridisation PCRfragments were amplified from genomic DNA of pure cultures or plasmid DNA of the
algal target-species by using the primer pairs listed in table 1. The PCR was carried
out for all primer combinations in a PCR-cycler from Eppendorf with the following
protocol: 5 min 94 oC, 2 min 94 oC, 1 min 54 oC, 3 min 72 oC, 35 cycles, 10 min 72
oC.
A 250 bp fragment was amplified from the S. cerevisiae TBP-gene with the primer
pair TBP-F (5’-ATG GCC GAT GAG GAA CGT TTA A-3’) and TBP-R-Biotin (5’-TTT
TCA GAT CTA ACC TGC ACC C- 3’) to serve as a positive control in later
experiments. The amplification of the PCR-fragment was done as follows: 5 min 94
oC,
1 min 94 oC, 1 min 52 oC, 1 min 72 oC, 35 cycles, 10 min 72 oC. Prior to the
hybridisation the PCR-fragment was purified with the QIAquick PCR purification kit
(Qiagen,
Hilden,
Germany).
The
amount
of
DNA
was
determined
spectophotometrically by measuring the optical density at 260 nm with a photometer
(Eppendorf).
Microchip-Fabrication. Oligonucleotide probes for microarray printing were obtained
from Thermo Hybaid, Interactiva Division (Ulm, Germany) with a C6/MMT Aminolink
at the 5’-end of the molecule. The manufacturing of the slides and the printing of the
oligonuclotides onto the chip was carried out by PicoRapid GmbH (Bremen). After the
printing procedure the DNA microchips were stored at –20 oC.
Hybridisation of 18S PCR-fragements to DNA-microchips. The hybridisation
cocktail contained biotinylated 18S PCR-fragment at a final concentration of 20 ng/µl
and a 250 bp PCR-fragment of S.cerevisiae TBP at a final concentration of 4 ng/µl in
hybridisation-buffer (1M NaCl/10 mM Tris, pH 8/ 0,005 % Triton X-100/ 1 mg/ml BSA/
0,1 µg/µl HS-DNA) at a total volume of 100 µl. Prior to the hybridisation a prehybridisation of the DNA-chips was carried out in hybridisation buffer for 60 min at
hybridisation temperature. After this the hybridisation cocktail was denatured for 5
min at 94 oC. Following the denaturation the hybridisation cocktail was pipetted
directly onto the DNA-microchip. The hybridisation was carried out under a coverslip
in a wet chamber at 58 oC for one hour. After hybridisation the DNA-microchips were
washed immediately for 15 min in 50 ml of buffer 1 (2x SSC/10 mM EDTA/0,05 %
SDS) and for additional 15 min in 50 ml buffer 2 (1 x SSC/10 mM EDTA).
Staining of the hybridised DNA-microchips. Hybridised biotinylated target DNA
was visualised by staining the DNA-microchips 30 min with Streptavidin-Cy5
in
hybridisation-buffer at a final concentration of 50 ng/ml.
Scanning and quantification of microarrays. Fluorescence images of the
hybridised DNA-microchips ware taken with the Genepix 4000B-Scanner (Axon
Instruments Inc., USA). The signal intensities have been quantified using the
GenePix 4.0-software (Axon Instruments Inc., USA). To quantify each single spot a
grid of individual circles defining the location of a spot was superimposed onto the
image. The mean signal intensity and the intensity of the local background area was
determined for each spot.
Results
Probe set. For subsequent DNA-chip experiments a set of 20 probes was chosen
that targets the 18S-DNA of the major picoplanktonic groups on the level of division,
class or genus. The probes were all known to work specific with dot-blots or FISH. A
list of the probes on the DNA-chip is given in table 2. In addition to the probes that
target phytoplankton, one probe was chosen that binds to a 250 bp PCR- fragment
from S. cerevisiae. The PCR-fragment is added to each hybridisation serving as an
external positive control to estimate the hybridisation efficiency. Another probe was
chosen to serve as a negative control, that did not have any match against a BLAST
search (Altschul et al., 1990).
Hybridisation results. The specificity of the probes on the chip was evaluated by
separate hybridisations of 18S- PCR-fragments that perfectly matched the probes on
the chip. For each probe one reference algae was chosen. The PCR-fragments were
either amplified from genomic DNA isolated from laboratory algal strains or from
picoplanktonic clones isolated in the FP5-project PICODIV. Since the binding loci of
the probes were distributed over the complete sequence, it was necessary to
hybridise the complete 18S-gene. The PCR-fragments had a size of 1800 bp
representing almost the complete 18S-gene.
This approach corresponded to
previous publications that reported accessing picoplankton diversity by analysing the
18S gene ( Díez et al., 2001; Moon-van der Staay et al., 2001). For the quantitative
analysis of the signal intensities, the setting of the PMT in the microarray-reader
Genepix 4000 B (Axon Instruments Inc., USA) was 750 at full laser-strength.
Subsequent to determining the signal to noise ratio, the signal of the positive control
was used for normalisation of the different hybridisations. The signal to noise ratio
was determined as described before by Loy et al., 2002. Evaluation of the chip with
the matching reference strains revealed a threshold value of 2.0 for a specific signal.
This observation is accordant to the previously mentioned publication (Loy et al.,
2002). In this context 12 out of 20 tested probes resulted in a specific signal with a
signal to noise ratio that was greater than the threshold of 2.0 for the perfectly
matching target DNA (Tab. 3). Only one probe appeared to be unspecific by binding
to several non target DNAs. However six probes did not result in a detectable signal
at all. This group of probes consisted of the probes Euk1209, Boli01, Hetero01,
Dino01, Chlo01, Pela01 and Pela02. It was not possible to determine a signal for
these probes even though the perfectly matching target was used for hybridisation.
Correlation of probe loci with signal intensities and secondary structure. The
previously described results show that not all of the probes, which have been
selected, resulted in equaly strong signals if they were hybridised to the matching
18S-DNA. The binding-loci have been correlated with the signal-intensities of the
hybridisation experiments and it appears that the majority of the probes that hardly
generate a signal is located all at a distance > 1000 bp from the 5’-End of the 18Sgene (Table 2). First of all it was tested if the integrity of the DNA was influenced in
the course of the hybridisation. Since the target DNA was labelled at the 5’-end of the
molecule by the use of a biotinylated primer during the PCR-amplification-step, it was
essential for a successful hybridisation that the PCR-fragment remained intact under
hybridisation conditions.
The integrity of the target DNA was determined on an
agarose gel after an incubation at hybridisation conditions. It appeared that the target
DNA was not severely fragmented (data not shown).
Steric hindrance. In previous publications (Loy et al., 2002) it was discussed that
dramatic differences in signal intensities of probes that target the same species might
be due to steric hindrance or secondary structures of the molecule. In order to
determine if the missing signals are caused by steric hindrance, hybridisation
experiments with two smaller PCR- fragments of a size of 900 bp, that add up to the
complete sequence have been carried out. It was possible to observe a signal for
Chlo01 and Euk1209 if the smaller fragments were used for hybridisation (Fig.2).
However Boli01, Dino01 and Hetero01 did not result in a significant signal even
though the smaller fragments have been used for hybridisation (Data not shown). In
addition, a hybridisation experiment was carried out with PCR-fragments labelled at
both 5’ends hybridised to the matching reverse complements of the probes Euk
1209, Chlo01, Boli01, Dino01 and Hetero01.
In this set up those probes were
located in a distance of max. 1000 bp from the labelled 5’-end. However it was still
not possible to observe a signal for the reverse complement of Euk1209, Boli01and
Chlo01. The signal for the reverse complement of Dino01 was only slightly increased,
but for the reverse complement of Hetero01 it was possible to detect a strong signal,
which was not possible to be observed before (Fig.3). These data indicate that it is
rather the secondary structure of the molecule than steric hindrances that influences
the binding of the target DNA to the probe immobilised on the chip surface.
Influence of secondary structure on probe binding. The secondary structure of
the 18S rRNA was theoretically determined (Fig.1). A consensus secondary structure
was assembled from the 18S genes of different algal classes and the location of the
probes in the secondary structure was specified. The probes Euk1209, Boli01,
Chlo01, Dino01 and Hetero01 are located in an area of the rRNA molecule that was
discussed in other publications to comprise a number of regions that are blocked for
probe binding (Behrens et al., 2003). The effect of the secondary structure on the
probe binding was studied in more detail for Chlo01. The binding site of probe
Chlo01 is located at approximately base 1100 (Fig.1). In this area the bases 612 to
617 are paired to the bases 1106 to 1111. It was determined whether Chlo01 results
in a signal if the target DNA was truncated for the bases that form the double strand.
Two truncated versions of the 18S-gene were generated by PCR-amplification and
hybridised to the DNA-chip. If the target DNA was truncated for the bases 1 to 566,
there was still the possibility to form a double strand in the area of the binding site of
Chlo01. Therefore it was not expected to observe a signal. In accordance with the
expectations it was not possible to detect a signal for Chlo01 if the target DNA was
truncated for bases 1 to 566. However if the target DNA was truncated for bases 1 to
899 the possibility to form a double strand in the area of the binding site of Chlo01
was taken away. Here it was possible to detect a signal for Chlo01 (Fig.2). The
hybridisation of the latter truncated version of the target molecule had also an effect
on the signal of Euk1209, which is located in a long stretch of a hairpin structure that
is formed by bases 1173 to 1283 paired to bases 1426 to 1470. It is not possible to
observe a signal for Euk1209 if the target DNA is complete or truncated for the bases
1 to 566. However it is possible to detect a signal, if the target molecule is truncated
for the bases 1 to 899, even though these bases are not directly involved in forming
the hairpin (Fig.2). In contrast it is not possible to observe a signal for Dino01, Boli01
or Hetero01 if the target DNA is truncated for 899 bases at the 5’-end of the
molecule.
Discussion
Array Design and specificity of the probes. In this study probes that initially had
been designed for other hybridisation techniques were evaluated for their use on a
DNA-chip for the identification of picoplankton. A total number of 20 probes that
target phytoplankton at different taxonomic levels was chosen for this purpose. The
specificity of the probes was evaluated by hybridisations of the DNA-chip with
perfectly matching 18S-DNAs of reference organisms for each probe on the chip. In
contrast to the results of other hybridisation techniques on the DNA-chip only 12 out
of 20 tested probes resulted in a specific signal if a threshold of 2.0 for the signal to
noise ratio was taken as a criterion for a significant signal. However one probe
appeared to be unspecific and seven probes did not result in any signal at all. Even
though the vast majority of the probes that resulted in a signal appeared to be
specific, some of the probes e.g. Bathy 01 or Ostero 01 resulted in a signal to noise
ratio that was close to the threshold of 2.0 if they were hybridised to a non target
organism. These cross interactions could lead to a misinterpretation if complex
samples have to be analysed. However this problem could be avoided by the use of
multiple probes for one target organism on the chip. This approach was implemented
partially on our chip. The chip contained two probes for some targets. However in
accordance to other publications (Milner et al., 1997; Southern et al., 1999; Loy et al.,
2002) we found tremendous differences in the signal intensities of probes that target
the same species. It was possible to observe a significant signal for Boli02, Chlo01
and DinoE-12. However the corresponding probes Boli01, Chlo1 and Dino01,
targeting the same species, displayed no signal at all even though it was shown
before that they work for other hybridisation techniques (Simon et al., Simon et al.,
John et al.,2003).
Differences to other hybridisation techniques. Here we present that probes that
all have been developed for other hybridisation techniques like dot blot hybridisation
or FISH do not all work equally well if they are used for DNA-chip hybridisation. The
differences between results generated by other hybridisation techniques to the
results presented here could be explained by the nature of the target. If the probes
are used for in situ hybridisation the system contains proteins that bind to the target
nucleic acid and influence thereby the secondary structure of the nucleic acid. As a
consequence it is conceivable that binding sites that are accessible in situ are
inaccessible if the pure nucleic acid without proteins is hybridised to a DNA-chip and
vice versa. In a recent study a map of the in situ accessibility of the 18S rRNA of S.
cerevisiae was pictured (Behrens et al., 2003). We compared our data with the data
shown in this publication. We found that the overall trend is similar to the
observations that we made with the microarray. Loops that were described for having
poor signals appeared to have poor signals on the microarray too. Nevertheless this
is just a trend that gives only a hint of what might be real. It is difficult to compare
results generated with two different techniques from organisms which are only
distantly related.
Correlation of signal intensities with binding loci. The correlation of signal
intensities with the binding loci of the probes indicate that the observed differences
are linked to the binding loci in the target DNA. The binding sites of the majority of the
probes that resulted in a specific signal were located in an area up to approximately
1000 bases of the target DNA. Previous publications discuss (Loy et al., 2002) that
steric hindrances could be the reason for a missing signal. In this study it was tested
whether the problem of missing signals could be overcome by avoiding steric
hindrance by the use of two smaller fragments of a size of 900 bp for the
hybridisation. However it turned out that a hybridisation of the smaller fragments only
resulted in signals for Euk1209 and Chlo01. The other probes still did not result in
significant signals. The possibility of steric hindrance was tested in a second
approach using the reverse complement of probes Euk1209, Boli01, Chlo01, Dino01,
and Hetero01 on the DNA chip. In contrast to their forward counterparts the probes
had binding sites that are located max. 1000 bp from the 5’end of the target DNA. In
this set up a strong signal for Hetero01 could be observed, indicating that for
Hetero01 steric hindrance was the reason for the missing signal. However it was not
possible to observe a significant increase of the probe signals for Euk1209, Boli01,
Chlo01 and Dino01. These results indicate that steric hindrance is not the major
reason for the missing signals of these probes. It is rather feasible that the secondary
structure has a major impact on their hybridisation capacity. The probes are all
located further downstream than 1000 bases from the 5’-end of the molecule. In the
previously mentioned publication of Behrens et al., 2003 it was indicated that the
area downstream of 1000 bases in the molecule harbours more regions that are not
or only poorly accessible to oligonuclotide probes in situ (Behrens et al., 2003).
Therefore it is potentially more difficult for probes to access the binding site in this
area than in the remaining part of the molecule, where the determined secondary
structure of the algal 18S rRNA were only one region was found that is not accessible
to oligonucleotide probes.
The binding of Chlo01 and Euk1209 is influenced by the secondary structure.
The potential influence of the secondary structure on the binding was further studied
by a hybridisation of different truncated versions of the target DNA to the chip. Two
truncated versions of the target molecule were hybridised to the DNA-chip. It
appeared that the use of the different truncated versions did not have an effect on the
probes DinoB, Boli01 and Hetero01. And if the target molecule was truncated for
bases 1 to 592 it was also not possible to observe a hybridisation signal for Chlo01
and Euk1209. This is consistent with the determined secondary structure that
displays paired bases at the binding site of Chlo1 and a long hairpin at the binding
site of Euk1209. The possibility to form the described secondary structures remained
unaffected if the target molecule was truncated for bases 1 to 592. However if the
target DNA truncated for bases 1 to 899 was used for hybridisation it was possible to
measure a signal for the probes Euk1209 and Chlo01. We hypothesise that this
truncated version of the target molecule is hampered for the formation of a secondary
structure at the binding site of Chlo01, leaving it possibly free for a hybridisation to
the probe. The secondary structure at the binding site of Euk1209 is not directly
influenced by the lack of bases 1 to 899 but it might be that in that area it is altered in
the truncated version of the target DNA or that in the complete rRNA molecule the
the truncated part forms a higher order structure with the part that harbours the
binding site of Euk1209.
Recapitulating it was possible to developed a DNA-chip that targets the 18S rRNA of
phytoplankton respectively picoplankton at different taxonomic levels. A set of 20
probes that were known to work for other hybridisation techniques was chosen to be
tested for the use on DNA-chips. It appeared that the majority of those probes that
were located in an area up to 1000 bp in the target molecule resulted in a significant
signal. While those probes that were located further downstream in the molecule did
not result in a signal at all. Our results strongly indicate that the secondary structure
of the 18S rRNA has a major impact on the hybridisation efficiency of a probe. Stable
secondary structures likely make a probe binding site inaccessible. This problem has
been addressed before for prokaryotes (Fuchs et al., 1998 and Behrens et al., 2003).
It is published that it is possible to overcome problems caused by higher-order
structures by adding unlabeled helper oligonucleotide that bind in close proximity to
the probe. In this respect the helper oligonucleotide has a synergistic effect for the
probe binding (Fuchs et al., 2000). However the use of helper oligonucleotides is a
good solution for single probe methods like FISH, but the use of helper
oligonucleotides for a larger number of probes like it is the situation on DNA-chips
would be rather complex. Our results indicate that for the algal 18S rRNA the
problem of higher-order structures that prevent probe binding can be overcome by
placing the probes in an area up to a distance of 1000 bp from the 5’-end of the
molecule.
Acknowledgements
We would like to thank Dr. Klaus Valentin for providing the clones of the
Bolidophyceae and the Chlorophyceae and Helga Mehl for excellent technical
assistance. This work was supported by a grant of the EU for the FP5-project
PICODIV (Contract EVK3-CT-1999-00021).
References
Altschul, S.F., W. Gisch, W. Miller, E.W. Myers & D.J. Lipman (1990): Basic local
alingment search tool. - J. Mol. Bio. 215: 403-410
Andersen, R.A., G.W. Saunders, M.P. Paskind, and J.P. Sexton (1993).
Ultrastructure and 18SrRNA gene sequence for Pelagomonas calceolata gen. et sp.
nov. and the description of a new algal class, the Pelagophyceae classis nov. J.
Phycol. 29: 701-715
Behrens S., C. Rühland, J. Inácio, H. Huber, A. Fonseca, I. Spencer-Martins, B.M.
Fuchs and R. Amann (2003). In Situ Accessibility of Small-Subunit rRNA of Members
of the Domains Bacteria, Archea, and Eucarya to Cy3-Labeled Oligonucleotide
Probes. Appl. Environ. Microbiol. 69 : 1748-1758
Campbell, L., H.A. Nolla, and D. Vaulot (1994). The importance of Prochlorococcus
to community structure in the central North Pacific Ocean. Limnol. Oceanogr. 39:
954-961
DeLong, E.F., G.S. Wickham & N.R. Pace (1989): Phylogenetic stains: ribosomal
RNA-based probes for the identification of single cells. – Science 243: 1360-1363
DeRisi, J.L., V. R. Iyer & P.O. Brown (1997): Exploring the metabolic and genetic
control of gene expression on a genomic scale. – Science 278: 680-686
Díez, B., C. Pedrós-Alió & R. Massana (2001): Study of genetic Diversity of
eukaryotic picoplankton in different oceanic regions by small-subunit rRNA Gene
Cloning and Sequencing. – Appl. Environ. Microbiol. 67:2942-2951.
Fuchs, B.M., G. Wallner, W. Beisker, I. Schwippl, W. Ludwig and R. Amann (1998).
Flow Cytometric Analysis of the In Situ Accessibility of Escherichia coli 16S rRNA for
Fluorescently Labeled Oligonucleotide Probes. Appl. Environ. Microbiol. 64 : 49734982
Fuchs, B.M., F.O. Glöckner, J. Wulf & R. Amann (2000): Unlabeled Helper
Oligonucleotides Increase the In Situ Accessibility to 16S rRNA of Fluorescently
Labeled Oligonucleotides Probes. – Appl. Environ. Microbiol. 66: 3603-3607
Giovannoni, S. J., T.B. Britschgi, C.L. Moyer & K.G. Field (1990): Genetic diversity in
Sargasso Sea bacterioplankton. - Nature 345: 60-63
Guillou, L., M.J. Chétiennot-Dinet, L.K. Medlin, H. Claustre, S. Loiseaux-de Goer & D.
Vaulot (1999) : Bolidomonas : a new genus with two species belonging to new algal
class, the Bolidophyceae (Heterokonta). – J. Phycol. 35: 368-381
Guillou, L., Moon-van-der-Staay, S.Y., Claustre, H., Partensky, F. and Vaulot, D.
(1999). Diversity and abundance of Bolidophyceae (Heterokonta) in two oceanic
regions. Appl. Environ. Microbiol., 65, 4528-4536.
Guschin, D.Y., B.K. Mobarry, D. Proudnikov, D.A. Stahl, B.E. Rittmann & A. D.
Mirzabekov (1997): Oligonucleotide microchips as genosensors for determinative and
environmental studies in microbiology. – Appl. Environ. Microbiol. 63: 2397-2402
John, U., A. Cembella, C. Hummert, M. Elbrächter, R. Groben and L. Medlin (2003).
Discrimination of the toxigenic dinoflagellates Alexandrium tamarense and A.
ostenfeldii in co-occurring natural populations from Scottish coastal waters. Eur.J.
Phycol. 38: 25-40
Lange, M., L. Guillou, D. Vaulot, N. Simon, R.I. Amann, W. Ludwig & L.K. Medlin
(1996): Identification of the class Prymnesiophyceae and the genus Pheocystis with
ribosomal RNA-target nucleic acid probes detected by flow cytometry. – J. Phycol.
32: 858-868
Li , W.K.W. (1994) Primary productivity of prochlorophytes, cyanobacteria, and
eucaryotic ultraphytoplankton: Measurements from flow cytometric sorting. Limnol.
Oceanogr. 39: 169-175
Lim, E.L., L.A. Amaral, D.A. Caron & E.F. DeLong (1993) : Application of rRNAbased probes for observing marine nanoplanktonic protists. – Appl. Environ.
Microbiol. 59:1647-1655
Lim, E.L., M.R. Dennett & D.A. Caron (1999): The ecology of Paraphysomonas
imperforata based on studies employing oligonucleotide probe identification on
coastal water samples and enrichment cultures. – Limnol. Oceanogr. 44: 37-51
Lockhart, D.J., H. Dong, M.C. Byrne, M.T. Follettie, M.V. Gallo, M.S. Chee, M.
Mittmann, C. Wang, M. Kobayashi, H. Horton & E.L. Brown (1996): Expression
monitoring by hybridization to high-density oligonucleotide arrays. – Nat. Biotechnol.
14:1675-1680
López-Garcia, P., F. Rodriguez-Valera, C. Pedros-Alio & D. Moreira (2001):
Unexpected diversity of small eukaryotes in deep-sea Antarctic plankton. – Nature
409: 603-607
Loy, A., A. Lehner, N. Lee, J. Adamczyk, H. Meier, J. Ernst, K.H. Schleifer, and M.
Wagner (2002). Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of
All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment. Appl.
Environ. Microbiol. 68: 5064-5081
Maidak, B.L., J.R. Cole, T.,G, Lilburn, C.T.J. Parker, P.R. Saxman, R.J. Farris, G.M.
Garrity, G.J. Olson, T.M. Schmidt & J.M. Tiedje (2001): The RDP-II (Ribosomal
Database Project). – Nucleic. Acids. Res. 29: 173-174
Medlin, L., H.J. Elwood, S. Stickel & M.L. Sogin (1988): The characterization of
enzymatically amplified eukaryotic 16S-like rRNA-coding regions. - Gene 71:491-499
Miller, P.E. & C.A. Scholin (1998): Identification and enumeration of cultured and wild
Pseudo-Nitzschia (Bacillariophyceae) using species-specific LSU rRNA-targeted
fluorescent probes and filter based whole cell hybridization. – J. Phycol. 34: 371-382
Milner, N., K.U. Mir, and E. M. Southern (1997). Selecting effective antisense
reagents on combinatorial oligonucleotide arrays. Nat. Biotechnol. 15: 537-541
Moon-van der Staay, S.Y., R. De Wachter & D. Vaulot (2001): Oceanic 18S rDNA
sequences from picoplankton reveal unsuspected eukaryotic diversity. -Nature 409:
607-610
Partensky, F., W.R. Hess, and D. Vaulot (1999). Prochlorococcus, a marine
photosynthetic prokaryote of global significance. Microb. Mol. Biol. Rev. 63: 106-127
Simon, N., LeBot, N., Marie, D., Partensky, F. and Vaulot, D. (1995). Fluorescent in
situ hybridization with rRNA-targeted oligonucleotide probes to identify small
phytoplankton by flow cytometry. Appl. Environ. Microbiol., 61, 2506-2513.
Simon, N., L. Campbell, E. Ornolfsdottir, R. Groben, L. Guillou, M. Lange & L.K.
Medlin (2000): Oligonucleotide probes for the identification of three algal groups by
dot blot and fluorescent whole-cell hybridization. – J. Eukaryot. Microbiol. 47: 76-84.
Southern, E., K. Mir, and M. Shchepinov (1999). Molecular interactions on
microarrays. Nat. Genet. 21 : 5-9
Table 1:
a.
b..
Primer
Sequence
Reference
1F-Biotin
690 F-Biotin
528F-Biotin
1528R
AAC CTG GTT GAT CCT GCC AGT
TCAGAGGTGAAATTCTTGGAT
GCGGTAATTCCAGCTCCAA
TGATCCTTCTGCAGGTTCACCTAC
Medlin et al., 1988
Medlin et al., 1988
Medlin et al., 1988
Medlin et al., 1988
1F-Biotin/1528R
1800 bp
528F-Biotin/1528R
1200 bp
690F/1528R
900 bp
Table 1.: a. Primer used for the amplification of PCR fragments for subsequent hybridisation. b. Primer combinations used to amplify truncated versions of 18S gene.
Table 2.:
Probe
Target
Sequence
Loci in 18S
Reference
Euk1209
All Eukaryotes
GGGCATCACAGACCTG
1430
Lim et al., 1993
Chlo 01
Chlorphyta
GCTCCACGCCTGGTGGTG
1100
Simon et al., 1995
Chlo 02
Chlorophyta
CTTCGAGCCCCCAACTTT
967
Simon et al., 2000
Boli 01
Bolidophyceae
CAGTCTGATTGAACTGCGT
1598
Guillou et al.,1999
Boli 02
Bolidophyceae
TACCTAGGTACGCAAACC
841
Guillou et al.,1999
Hetero 01
Stramenopiles
ACGACTTCACCTTCCTCT
1747
Eller & Medlin
Prym 01
Prymnesiophyta
ACATCCCTGGCAAATGCT
940
Lange et al., 1996
Prym 02
Prymnesiophyta
GGAATACGAGTGCCCCTGAC
871
Simon et al., 2000
Prym 03
Prymnesiophyta
GTCAGATTCGGGCAATTT
435
Eller & Medlin
Dino1
Dinophyta
CCTCAAACTTCCTTGCITTA
1392
John et al., 2003
Dino E-12
Dinophyta
CGGAAGCTGATAGGTCAGAA
305
Groben, John
Pela 01
Pelagophyta
ACGTCCTTGTTCGACGCT
931
Simon et al., 2000
Pela 02
Pelagophyceae Clade
GCAACAATCAATCCCAATC
Pras 04
Prasinophyceae
CGTAAGCCCGCTTTGAAC
747
Not
Bathy 01
Bathycoccus
ACTCCATGTCTCAGCGTT
636
Not
Micro 01
Micromonas
AATGGAACACCGCCGGCG
245
Not
Ostreo 01
Ostreococcus
CCTCCTCACCAGGAAGCT
609
Not
Crypto B
Cryptophyta
ACGGCCCCAACTGTCCCT
820
This work
NS 03
New Stramenopiles Clade 3
ATTACCTTGGCCTCCAAC
850
Massana
NS 04
New Stramenopiles Clade 4
TACTTCGGTCTGCAAACC
847
Massana
Positive control
S.cerevisiae
ATGGCCGATGAGGAACGT
-
This work
TCCCCCGGGTATGGCCGC
-
This work
Negative control
Simon et al., 2000
Tab. 2. : Probelist. In this table probes and sequences are listed that were spotted on the
DNA-chip.
Table 3 :
Probe
Signal/Noise Value
Target
Highest Signal/NoiseValue
Non Target
Euk 1209
Chlo 01
Chlo 02
Hetero 01
Boli 01
Boli 02
Prym 01
Prym 02
Prym 03
Pela 01
Pela 02
Dino 1
Dino E-12
NS 03
NS 04
Pras 04
Bathy 01
Micro 01
Ostreo 01
Crypto B
1,44
1,28
6,47
1,00
0,97
3,36
2,98
3,88
1,54
1,00
1,12
0,99
1,04
1,33
1,2
1,2
1,1
1,13
42,46
2,0
7,59
8,3
5,98
2,85
4,41
12,9
1,17
1,82
5,46
1,03
1,03
1,87
1,5
1,99
1,42
Table 3.: Signal / Noise ratios for target DNAs and non target DNAs. For the
non target DNAs the highest observed value is displayed.
Figure 1:
Figure 1.: Consensus secondary structure of the 18S rRNA. The binding sites of probes are
displayed as red lines for those probes that resulted in no signals and in green lines for those
probes that resulted in signal to noise ratios that were above 2.0. Purple lines display the
primer that have been used to amplify the target DNA.
Figure 2:
35
30
25
20
15
10
5
0
1F/1528R
528F/1528R
690F/1528R
Eu
k
12
C 09
hl
o
C 01
hl
H o0
et
er 2
o
Bo 01
li
Bo 01
l
Pr i 02
ym
Pr 01
ym
Pr 02
ym
Pe 03
la
Pe 01
la
0
D 2
D in o
in
o B
E1
N 2
S
0
N 3
S
Pr 04
a
Ba s 0
th 4
M y 01
ic
O ro 0
st
re 1
o
C 01
ry
pt
o
B
N
K
Signal / Noise
He001005-53 (Chlorophyceae)
Probes
Fig. 2.: Hybridisation of three different fragments of the 18S-DNA amplified from clone
HE001005-53, a Chlorophyceae, isolated in the course of PICODIV.
Figure 3 :
He001005-51
HE001005-53
U
K
E
E
N
K
N
S
P
R 4
A
S
C
R
YP 04
C TO
R
YP B
TO
A
12
1 2 09
09
C RK
H
C LO
H
LO 01
01
C RK
H
LO
P
R 02
Y
M
P
R 01
Y
M
P
R 02
Y
M
0
D 3
IN
O
D
in
o B
B
D
IN RK
O
E
B 12
B OL
I0
O
LI
1
01
R
K
B
H OL
H ET I 0 2
E
E
TE R
R O0
O
1
01
R
P
EL K
A
01
He001005-127
U
K
Signal / Noise
50
45
40
35
30
25
20
15
10
5
0
Probes
Fig. 3.: Quantitative analysis of signal intensities measured after a hybridisation of a
bolidophyceae (He001005-51), a chlorophyceae (He001005-53) and a dinophyceae
(He001005-127) to a DNA-chip that contained the indicated probes.