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A novel design of whole-genome microarray probes for Saccharomyces cerevisiae which minimizes cross-hybridization.

Talla E, Tekaia F, Brino L, Dujon B - BMC Genomics (2003)

Bottom Line: We present here a novel design of Saccharomyces cerevisiae microarrays based on a refined annotation of the genome and with the aim of reducing cross-hybridization between related sequences.The sequence of each gene was compared against the entire yeast genome and optimal sub-segments giving no predicted cross-hybridization were selected.The OliD program is available from authors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Pasteur, Unité de Génétique Moléculaire des Levures (URA 2171 CNRS, UFR 927 Université PM Curie), 25 rue du Docteur Roux, F-75724 Paris cedex 15, France. etalla@pasteur.fr

ABSTRACT

Background: Numerous DNA microarray hybridization experiments have been performed in yeast over the last years using either synthetic oligonucleotides or PCR-amplified coding sequences as probes. The design and quality of the microarray probes are of critical importance for hybridization experiments as well as subsequent analysis of the data.

Results: We present here a novel design of Saccharomyces cerevisiae microarrays based on a refined annotation of the genome and with the aim of reducing cross-hybridization between related sequences. An effort was made to design probes of similar lengths, preferably located in the 3'-end of reading frames. The sequence of each gene was compared against the entire yeast genome and optimal sub-segments giving no predicted cross-hybridization were selected. A total of 5660 novel probes (more than 97% of the yeast genes) were designed. For the remaining 143 genes, cross-hybridization was unavoidable. Using a set of 18 deletant strains, we have experimentally validated our cross-hybridization procedure. Sensitivity, reproducibility and dynamic range of these new microarrays have been measured. Based on this experience, we have written a novel program to design long oligonucleotides for microarray hybridizations of complete genome sequences.

Conclusions: A validated procedure to predict cross-hybridization in microarray probe design was defined in this work. Subsequently, a novel Saccharomyces cerevisiae microarray (which minimizes cross-hybridization) was designed and constructed. Arrays are available at Eurogentec S. A. Finally, we propose a novel design program, OliD, which allows automatic oligonucleotide design for microarrays. The OliD program is available from authors.

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Related in: MedlinePlus

Effects of probe size, GC probe content and position relative to stop codon on signal intensities. Cy3- and Cy5-labelled cDNA targets were prepared from the total RNA isolated from the wild-type strain BY4742 and Δymr011w, respectively. Hybridization was performed as described in "Methods" with microarray from Batch616. For the Cy5 channel, normalized signals of each spot were computed as a function of probe size (panel A), GC probe content (panel B) and the distance from 3'end of the probe to stop codon of the CDS (panel C). Panel A includes PCR probes only, panels B and C include both PCR and oligonucleotide probes. Red curves represent running averages of the signal intensities.
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Figure 3: Effects of probe size, GC probe content and position relative to stop codon on signal intensities. Cy3- and Cy5-labelled cDNA targets were prepared from the total RNA isolated from the wild-type strain BY4742 and Δymr011w, respectively. Hybridization was performed as described in "Methods" with microarray from Batch616. For the Cy5 channel, normalized signals of each spot were computed as a function of probe size (panel A), GC probe content (panel B) and the distance from 3'end of the probe to stop codon of the CDS (panel C). Panel A includes PCR probes only, panels B and C include both PCR and oligonucleotide probes. Red curves represent running averages of the signal intensities.

Mentions: We have examined the effects of probe size, GC content, and position relative to stop codon on signal strength (case of Cy5 channel). Results of hybridization experiment with Δymr011w strain are shown by Figure 3. The probe size (300–700 bp) has no significant effect on signal although the running average of the intensities ranges from 2039 to 4145. The same is essentially true for the distance between the probe and the stop codon (except for very high figures (>1000 nt)). On the contrary, the GC probe composition shows some influence on intensities, especially for lower GC as could be expected. As can be seen, the signal intensity increases with the GC probe content with a maximun at 44 to 46% GC. Note, however, the large distribution of values over the entire composition range. Similar results were obtained for the Cy3 channel (data not shown). Moreover, the figure observed in Δymr011w hybridization is the same in other hybridization experiments (data not shown).


A novel design of whole-genome microarray probes for Saccharomyces cerevisiae which minimizes cross-hybridization.

Talla E, Tekaia F, Brino L, Dujon B - BMC Genomics (2003)

Effects of probe size, GC probe content and position relative to stop codon on signal intensities. Cy3- and Cy5-labelled cDNA targets were prepared from the total RNA isolated from the wild-type strain BY4742 and Δymr011w, respectively. Hybridization was performed as described in "Methods" with microarray from Batch616. For the Cy5 channel, normalized signals of each spot were computed as a function of probe size (panel A), GC probe content (panel B) and the distance from 3'end of the probe to stop codon of the CDS (panel C). Panel A includes PCR probes only, panels B and C include both PCR and oligonucleotide probes. Red curves represent running averages of the signal intensities.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC239980&req=5

Figure 3: Effects of probe size, GC probe content and position relative to stop codon on signal intensities. Cy3- and Cy5-labelled cDNA targets were prepared from the total RNA isolated from the wild-type strain BY4742 and Δymr011w, respectively. Hybridization was performed as described in "Methods" with microarray from Batch616. For the Cy5 channel, normalized signals of each spot were computed as a function of probe size (panel A), GC probe content (panel B) and the distance from 3'end of the probe to stop codon of the CDS (panel C). Panel A includes PCR probes only, panels B and C include both PCR and oligonucleotide probes. Red curves represent running averages of the signal intensities.
Mentions: We have examined the effects of probe size, GC content, and position relative to stop codon on signal strength (case of Cy5 channel). Results of hybridization experiment with Δymr011w strain are shown by Figure 3. The probe size (300–700 bp) has no significant effect on signal although the running average of the intensities ranges from 2039 to 4145. The same is essentially true for the distance between the probe and the stop codon (except for very high figures (>1000 nt)). On the contrary, the GC probe composition shows some influence on intensities, especially for lower GC as could be expected. As can be seen, the signal intensity increases with the GC probe content with a maximun at 44 to 46% GC. Note, however, the large distribution of values over the entire composition range. Similar results were obtained for the Cy3 channel (data not shown). Moreover, the figure observed in Δymr011w hybridization is the same in other hybridization experiments (data not shown).

Bottom Line: We present here a novel design of Saccharomyces cerevisiae microarrays based on a refined annotation of the genome and with the aim of reducing cross-hybridization between related sequences.The sequence of each gene was compared against the entire yeast genome and optimal sub-segments giving no predicted cross-hybridization were selected.The OliD program is available from authors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut Pasteur, Unité de Génétique Moléculaire des Levures (URA 2171 CNRS, UFR 927 Université PM Curie), 25 rue du Docteur Roux, F-75724 Paris cedex 15, France. etalla@pasteur.fr

ABSTRACT

Background: Numerous DNA microarray hybridization experiments have been performed in yeast over the last years using either synthetic oligonucleotides or PCR-amplified coding sequences as probes. The design and quality of the microarray probes are of critical importance for hybridization experiments as well as subsequent analysis of the data.

Results: We present here a novel design of Saccharomyces cerevisiae microarrays based on a refined annotation of the genome and with the aim of reducing cross-hybridization between related sequences. An effort was made to design probes of similar lengths, preferably located in the 3'-end of reading frames. The sequence of each gene was compared against the entire yeast genome and optimal sub-segments giving no predicted cross-hybridization were selected. A total of 5660 novel probes (more than 97% of the yeast genes) were designed. For the remaining 143 genes, cross-hybridization was unavoidable. Using a set of 18 deletant strains, we have experimentally validated our cross-hybridization procedure. Sensitivity, reproducibility and dynamic range of these new microarrays have been measured. Based on this experience, we have written a novel program to design long oligonucleotides for microarray hybridizations of complete genome sequences.

Conclusions: A validated procedure to predict cross-hybridization in microarray probe design was defined in this work. Subsequently, a novel Saccharomyces cerevisiae microarray (which minimizes cross-hybridization) was designed and constructed. Arrays are available at Eurogentec S. A. Finally, we propose a novel design program, OliD, which allows automatic oligonucleotide design for microarrays. The OliD program is available from authors.

Show MeSH
Related in: MedlinePlus