Poster

1
Development and Evaluation of a Comprehensive
Functional Gene array for Environmental Studies
Zhili He1,2, C. W. Schadt2, T. Gentry2, J.
Liebich3, S.C. Song2, X. Li4, and J. Zhou 1,2
1The University of Oklahoma, Norman, OK, 2Oak
Ridge National Laboratory, Oak Ridge, TN,
3Forschungszentrum Julich GmbH, Julich, Germany,
4Perkin Elmer Life and Analyetical Sciences,
Boston, MA
N125
http//ieg.ou.edu/
RESULTS
ABSTRACT

• For oligo targets, there were three false
  positives and two false negatives, and for
  PCR-amplicon targets, four false positives and no
  false negatives observed (Table 5).
• Possible reasons include (i) First, the amounts
  of some oligonucleotides or PCR-amplicons applied
  to the array was too high or too low (ii) Probe
  design criteria used were not specific enough for
  excluding all non-specific probes, and that some
  additional criteria may need to be considered
  (iii) an optimization of hybridization conditions
  may improve probe specificity (iv) there may be
  errors in probe or/and gene sequences.
• To tackle the problem of false positives,
  relative comparisons are needed.

To detect and monitor functions of microbial
organisms in their environments, functional gene
arrays (FGAs) have been used as a promising and
powerful tool. In this study, we have constructed
the second generation of FGA, called FGA2.0 that
contains 23,843 oligonucleotide (50mer) probes
and covers more than 10,000 sequences of targeted
genes, which are involved in nitrogen, carbon,
sulfur cycling and metabolism, metal reduction
and resistance, and organic contaminant
degradation. Several new strategies have been
implemented in probe design, array construction
and data analysis. Gene sequences were
automatically retrieved by key words. A newly
developed oligonucleotide design program
CommOligo was used to select gene-specific and
group-specific probes, and multiple probes were
designed for each gene sequence or each group of
highly homologous sequences. All designed
oligonucleotides were verified and output in a
96-well format for direct order placement of
oligonucleotide synthesis. To ensure the array
specificity, the array has been systematically
evaluated using different targets and
environmental samples. The results demonstrate
that such an array can provide specific analysis
of microbial communities in a rapid,
high-through-put and cost-effective fashion.
Fig. 1 Major steps for construction of a
comprehensive 50mer oligo functional gene array.
CommOligo is the core program to select
gene-specific and group-specific oligonucleotide
probes. GeneDownloader, ProbeChecker, and
PlateProducer were Perl scripts to pre-process
gene sequences or post-process oligonucleotide
probes.

• 15.2 probes target carbon metabolism genes
• 22.2 probe target the genes involved in
  nitrogen cycling
• 6.8 probes for sulfur reduction genes
• 3.6 probes for methane reduction and oxidation
• 19.0 probes target genes involved in metal
  reduction and resistance
• 34.0 probes target genes involved in
  degradation of organic compounds

Fig. 5 Relative signal intensities and SNR
values detected by probe A, B and C for
PCR-amplicon targets.

• Signal intensities for probe B and C were
  normalized with probe A (100), and there were
  14, 12 and 10 probe A, B, and C, respectively
  (Table S2 and Table S3).
• The average of relative signal intensities for
  probe A, B and C were 100, 103.8, and 97.6,
  respectively, and similarly, the average of SNR
  values were 73.1, 67.2 and 65.3 for probe A, B,
  and C, respectively (Fig. 5).
• The results suggest that three probes performed
  similarly with known targets.

EXPERIMENTAL DESIGN
Fig. 3
Fig. 2
Oligonucleotide design and synthesis. A computer
program CommOligo (Li et al., 2005) was used to
design gene-specific and group-specific probes
based on the following criteria (i)
gene-specific probes lt90 sequence identity,
lt20-base continuous stretch, and gt-35 kcal/mol
free energy (ii) group-specific probes gt96
sequence identity, gt 35-base continuous stretch,
and lt-60 kcal/mol free energy (He et al., 2005a
Liebich et al., 2006). Each gene sequence or a
group of homologous sequences had up to three
probes. All verified probes were synthesized
without modification by MWG Biotech, Inc. (High
Point, NC) in a 96-well plate format with the
concentration of 100 pmol/µl. Oligonucleotide
target synthesis. 25 oligonucleotides were
synthesized as gene-specific and group-specific
targets to evaluate the FGA specificity (Table
1). 50 pg for each oligonucleotide was used for
hybridizations with a single target or a mixture
of multiple targets. Preparations of
PCR-generated targets. 17 target genes were
selected, and their PCR products (PCR-amplicons)
were obtained using gene-specific primers and
standard PCR methods (Table 2 and Table 3). Each
PCR product had a minimal length to cover all
available probes (1, 2 or/and 3 depending on
probes selected) on the array. DNA labeling and
hybridization. The PCR-amplicons were
fluorescently labeled by random priming using
Klenow fragment of DNA polymerase as described
previously (He et al., 2005b). Hybridization was
at 50oC with 50 formamide.
CONCLUSIONS

• An FGA2.0 has been constructed with more than
  23,000 oligos covering more than 10,000 gene
  sequences. To our knowledge, this is the most
  comprehensive FGA for environmental studies.
• To ensure the array specificity, several new
  features has been implemented in the probe
  design, and array construction.
• The FGA2.0 has been systematically evaluated
  using oligonucleotide and PCR-amplicon targets,
  and demonstrates that it can be used as a
  powerful tool for a rapid, high-through-put and
  cost-effective analysis of microbial communities.
• The array can be used to profile microbial
  community differences, to address specific
  questions and/or hypotheses related to microbial
  population dynamics, and analyses of functional
  gene expression in microbial communities.

• For gene-specific probes, Fig. 2 shows the
  distribution of maximal sequence identities (Fig.
  2A), maximal stretch lengths (Fig. 2B), or
  minimal free energy (Fig. 2C) with their
  non-targets. Most of the probes (70) had
  maximal sequence identities 7284, stretch
  lengths 1215 bases, and 0-30kcal/mol free
  energy.
• For group-specific probes, Fig. 3 shows the
  distribution of minimal sequence identities (Fig.
  3A), minimal stretch lengths (Fig. 3B), or
  minimal free energy (Fig. 3C) with their group
  members. Most of the probes (92) had maximal
  sequence identities 100, stretch lengths 4550
  bases, and free energy values of -65 kcal/mol or
  smaller.

REFERENCES
He Z, Wu L, Li X, Fields MW and Zhou J (2005a).
Appl. Environ. Microbiol. 713753-3760. He Z, Wu
L, Fields MW and Zhou J (2005b). Appl. Environ.
Microbiol. 71 5154-5162. Li X, He Z and Zhou J
(2005). Nucleic Acid Res. 33 6114-6123
(Co-first authors). Liebich J, Schadt CW, Chong
SC, He Z, Rhee SK and Zhou J (2006). Appl.
Environ. Microbiol. 721688-1691.

• FGA II design strategies
• 1. Using MSA to identify conserved regions for
  each functional gene.
• 2. Using experimentally established
  oligonucleotide design criteria and the novel
  software tool CommOligo.
• 3. Designing gene-specific and group-specific
  probes.
• 4. Multiple probes for each sequence or each
  group of sequences.

Fig. 4 The FGA was hybridized with a mixture of
15 synthesized oligonucleotide targets at 42oC,
45oC, 50oC and 60oC. Balancing probe sensitivity
and specificity, the optimal hybridization
temperature was determined to be 45-50oC with 50
formamide, which is generally consistent with our
previous results.
ACKNOWLEDGEMENTS
This research was funded by the U.S. Department
of Energy (Office of Biological and Environmental
Research, Office of Science) grants from the
Genomes To Life Program and ERSP Program.