H. Construction of a full-genome CDNA microarray for Synechocystis
(performed in collaboration with Rob Burnap, Oklahoma State University
BMC Genomics 03
1. Characteristics of the Synechocystis sp. PCC6803 Genome
| Total Length: |
3573470 bp (circular) |
| Total Number of ORFs: |
3168 ORFs |
| - on direct strand: |
1660 ORFs |
| - on complementary strand: |
1508 ORFs |
| Percent coding sequence: |
87.01% |
| Percent coding intergenic region: |
12.99% |
| Average length of gene: |
981.4 bp |
| ORFs longer than 2000 bp |
2907 |
| ORFs shorter than 2000 bp |
261 |
| %C+G |
47.72 |
2. Global mRNA Expression ProfilingWith the
growing availability of complete genomic sequences for scores of prokaryotic organisms
and for several eukaryotes, efforts have turned to the development of experimental
approaches to measure transcription from a global perspective. DNA microarrays
have emerged as a particularly effective tool for genome-wide transcript profiling,
especially for studies examining eukaryotic organisms. Information on the temporal
patterns of accumulation and disappearance of transcripts for specific groups
of genes is suggestive of cellular programs orchestrating these changes [3, 10,
17, 18, 21] . Further developments in the biology now combine the power of knock-out
mutations of known regulatory loci with microarray analysis to help elucidate
the signal circuitry giving rise to programmatic changes in gene expression [16].
The efforts of tabs like that of Blattner [15, 20] and that field have helped
make arrays accessible for many bacterial systems.
3. DNA MicroarraysDNA microarray technology uses
microscopic, high-density arrays of DNA target elements immobilized to solid surfaces
such as microscope slides. The DNA target elements typically represent specific
gene sequences or sub-sequences that hybridize to cognate sequences in samples
under investigation (see below). There are currently two major types of DNA microarrays:
oligonucleotide microarrays and DNA fragment microarrays. Oligonucleotide microarrays
generally utilize in situ oligonucleotide synthesis techniques that directly
build individual DNA targets on the surface of the array [2]. Such an approach
is epitomized by the proprietary manufacturing methods of the Affymetrix Corporation,
a successful, but extremely expensive, implementation. The other type of array,
DNA fragment microarray, was initially pioneered for yeast by Brown at Stanford
University [4] and for E. coli by Blattner at the University of Wisconsin
[17]. This approach is technically accessible and now has been accomplished for
Synechocystis sp. PCC6803. 4. Project Design & Execution
The primary objective of the project was to construct DNA microarrays
for global analysis of transcription in Synechocystis sp. PCC6803. To
this end, PCR products for all genes were generated and arrayed onto surface-modified
glass slides. For the most part, the PCR gene set was comprised of full-length
genes. In instances where the gene is too long to permit efficient PCR amplification
in high throughput mode (>2 kb), only the 5' portion was amplified. The amplified
genes and gene fragments were flanked by 'adaptamers' that were introduced by
utilizing bipartite PCR primers consisting of both gene-specific and engineered
sequences [15]. The use of bipartite primers containing both gene-specific and
engineered sequences represents a two-stage amplification strategy (Figure 2)
that enables several downstream manipulations of the initially amplified gene-set
derived from genomic Synechocystis DNA as template. Most importantly,
the existence of common adaptamers on every member of the gene set permits subsequent
re-amplification of the entire gene set using common (not gene-specific) primers
that hybridize to the adaptamers that flank all members of the gene set produced
during the initial round of PCR amplification. Thus, the second round of amplification
used dilute aliquots of the first round PCR products as template. This affords
the ability to generate large amounts of PCR product without excessive depletion
of the gene-specific bipartite primer stocks. Additionally, the adaptamers contained
restriction sites to facilitate the directional cloning of the PCR products in
future studies.
2-stage amplification diagram
5. PCR primer design considerationsPrimers were designed
to amplify each of the 3168 genes present in the Synechocystis sp. PCC6803
genome as of May, 2002 [11]. The length of the PCR fragment was limited to 2kb
to promote efficient, high-yield amplification of the gene set. Using this criterion,
91% of the ORFs were full length, whereas the remaining 9% were truncated at the
3' ends. The primer design emulate that of the Blattner group [12,17], which incorporates
adaptamer sequences appended 5' to gene-specific portion of the bipartite primers.
A departure from the Blattner approach will be the modification of the adaptamer
sequence to allow re-amplification with a pair of common primers corresponding
to the two adaptamer sequences. We used ATG as the start codon for all amplified
ORFs irrespective of the actual codon in the genomic sequence, as developed by
Blattner. Likewise, TAA terminated every amplified coding sequence, even in the
case of the the 3' truncated versions of the long ORFs. These start and stop codons
were incorporated into the adaptamer sequences at the 5' and 3' ends of the amplified
coding sequence, respectively. The forward adaptamer sequence will consist of
5'-CTTGCTCTTCCATGNNNä.N-3' and the reverse adaptamer sequence 5'-GTTGCTCTTCGTTANNNä.N-3',
where N is a stretch of gene-specific nucleotides adding 20-25 gene-specific bases
to the 14 base common adaptamer sequence. The length of the gene-specific sequences
(NNN..N) were adjusted to achieve a melting temperature in the range of 68-70oC.
Sigma-Genosys performed the design and synthesis of the primers and supplied the
primers in a 96-well format as pairs of 96 forward and 96 reverse primers for
each 96 ORF sequences amplified.
Adaptamers
A portion of the adaptamer sequence corresponds to the non-palindromic 7-base
recognition sequence (GCTCTTC) for the restriction enzyme SapI, which
is a Type-IIS enzyme with an asymmetric recognition sequence and a cut site starting
one base from the 3' end of the recognition sequence and which leaves a 3-base
5' overhang upon cleavage. Introduction of the SapI sites in the adaptamers
at the 5' and 3' ends of the coding sequence, together with allowances to preserve
the open reading frame, allows for subsequent directional cloning and heterologous
expression of the amplified genes in future studies.
6. Amplification of gene setsPCR amplification of
the Synechocystis sp. PCC6803 gene-set was performed in two stages. The
objective of the proposed two-stage approach was to maximize the yield of PCR
products and to avoid depletion of the original stocks of bipartite PCR oligonucleotides.
These stocks will not only be used for amplification of the gene-set, but also
for the reverse transcription reaction generating the fluorescent hybridization
probe as discussed below. We feel it is important to maximize the yield to ensure
that the amount of DNA spotted onto the arrays is always saturating. The first
round of PCR generated a high fidelity 'master set' of gene fragments using Synechocystis
sp. PCC6803 genomic DNA as a template and the bipartite primers. The PCR reaction
was catalyzed by Invitrogen Pfu polymerase for maximizing fidelity. For
both stages of amplification, the PCR products were analyzed by gel electrophoresis
in high throughput fashion (96-lanes per gel).
7. Probe Generation & Microarray HybridizationMicroarray
experiments in prokaryotic organisms cannot utilize the poly(A) tails found in
eukaryotic mRNA; thus, alternatives to the highly successful methods employed
for generating fluorescent cDNA using oligo-dT to prime the reverse transcription
labeling reaction have to be devised. We have had moderate success using random
hexamers to prime the reaction. However, it will be necessary to generate fluorescent-labeled
cDNA in a manner that avoids labeling from highly abundant stable RNA species
(this tends to create a high level of non-specific background fluorescence that
diminishes the average signal-to-noise ratio of gene-specific hybridization signals
from the elements of the microarray). One alternative approach is to use the gene-specific
3' PCR primers manufactured to make the microarrays to also prime the cDNA reaction
generating the fluorescent replica of the mRNA population that is being tested.
Our preliminary results suggest that this increases the signal-to-noise ratio
several-fold. Thus, we wish to make allowances for the possibility that the users
will utilize the bipartite gene-specific primers each time an experiment is performed
with the microarrays. This approach also has potential downstream dividends, since
the introduction of adaptamer sequences allows for directional cloning of the
products as well as comparatively inexpensive modification of the adaptamers themselves
during secondary amplifications of the gene-set.
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