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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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A view of the microarray. The array is composed of 32 grids of 420 spots each. A total of ca. 200 empty spots are distributed through the array for background controls. Probe spots are deposited in close duplicates. A set of PCR products and synthetic oligonucleotides was selected as controls. These include scorecard kits (Amersham Biosciences) in grids 4, 8, 12, 16, 20, 24, 28, and 32; serial dilutions of the signal normalization luciferase gene (row 1 in grids 3, 7, 11, 15, 19, 23, 27, 31) for which control RNA spike (Promega) can be obtained; 10 long oligonucleotides covering YKL182w (6153 bp) and 9 long oligonucleotides covering YLR310c (4767 bp) ORFs as controls for reverse transcription efficiency; 10 intergenic regions, 10 intronic sequences, and mitochondrial genes, 20 non-monotonous trinucleotide repeats (72-mer oligonucleotides) and 4 serial dilutions (in grid 1, 4, 29 and 32) of the total genomic DNA from the wild-type strain S288c; 3 E. coli genes (tufA, aceF, kdtA) as negative controls; the LexA binding domain, the LacZ 5' and 3'end regions, the Pho4 binding domain, the Gal4 binding domain, the GFP, the TAP and GST as commonly used tags or reporter genes; Leu1, His5, and Ura4 from S. pombe, the humanCBF2 andc-myc genes, and the kanR gene as heterologous genes and markers. Printing buffer was deposited on the empty spots. The array shown results from hybridization with cDNA targets of total RNA isolated from wild-type BY4742 (Cy3-labelled) and Δydr225w (Cy5-labelled) strain.
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Figure 6: A view of the microarray. The array is composed of 32 grids of 420 spots each. A total of ca. 200 empty spots are distributed through the array for background controls. Probe spots are deposited in close duplicates. A set of PCR products and synthetic oligonucleotides was selected as controls. These include scorecard kits (Amersham Biosciences) in grids 4, 8, 12, 16, 20, 24, 28, and 32; serial dilutions of the signal normalization luciferase gene (row 1 in grids 3, 7, 11, 15, 19, 23, 27, 31) for which control RNA spike (Promega) can be obtained; 10 long oligonucleotides covering YKL182w (6153 bp) and 9 long oligonucleotides covering YLR310c (4767 bp) ORFs as controls for reverse transcription efficiency; 10 intergenic regions, 10 intronic sequences, and mitochondrial genes, 20 non-monotonous trinucleotide repeats (72-mer oligonucleotides) and 4 serial dilutions (in grid 1, 4, 29 and 32) of the total genomic DNA from the wild-type strain S288c; 3 E. coli genes (tufA, aceF, kdtA) as negative controls; the LexA binding domain, the LacZ 5' and 3'end regions, the Pho4 binding domain, the Gal4 binding domain, the GFP, the TAP and GST as commonly used tags or reporter genes; Leu1, His5, and Ura4 from S. pombe, the humanCBF2 andc-myc genes, and the kanR gene as heterologous genes and markers. Printing buffer was deposited on the empty spots. The array shown results from hybridization with cDNA targets of total RNA isolated from wild-type BY4742 (Cy3-labelled) and Δydr225w (Cy5-labelled) strain.

Mentions: Using the robot SDDC-2 (Engineering Services Inc., ESI), DNAs (PCR products, 71-mer oligonucleotides, and control genes) were deposited in close duplicates (1 fmol DNA of PCR product and 10 fmol DNA of long oligonucleotide) onto microarray aldehyde glass slides with 200-μm spacing between neighbouring spot centers. Subsequent microarray treatments were performed as recommended by the manufacturer (Telechem International). The microarray view is described in Figure 6. Note that batches 800, 400, and 616 (prepared successively in this order from November 2001 to February 2002) used in our hybridization experiments differ from the final ones described above, since the probes corresponding to the genes YBR145w, YDR225w, YEL033w, YGL147c, YGR148c, YJR148w, YMR011w, YMR169c, YMR170c, and YNL143c are present in the form of PCR products instead of 71-mer oligonucleotides.


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)

A view of the microarray. The array is composed of 32 grids of 420 spots each. A total of ca. 200 empty spots are distributed through the array for background controls. Probe spots are deposited in close duplicates. A set of PCR products and synthetic oligonucleotides was selected as controls. These include scorecard kits (Amersham Biosciences) in grids 4, 8, 12, 16, 20, 24, 28, and 32; serial dilutions of the signal normalization luciferase gene (row 1 in grids 3, 7, 11, 15, 19, 23, 27, 31) for which control RNA spike (Promega) can be obtained; 10 long oligonucleotides covering YKL182w (6153 bp) and 9 long oligonucleotides covering YLR310c (4767 bp) ORFs as controls for reverse transcription efficiency; 10 intergenic regions, 10 intronic sequences, and mitochondrial genes, 20 non-monotonous trinucleotide repeats (72-mer oligonucleotides) and 4 serial dilutions (in grid 1, 4, 29 and 32) of the total genomic DNA from the wild-type strain S288c; 3 E. coli genes (tufA, aceF, kdtA) as negative controls; the LexA binding domain, the LacZ 5' and 3'end regions, the Pho4 binding domain, the Gal4 binding domain, the GFP, the TAP and GST as commonly used tags or reporter genes; Leu1, His5, and Ura4 from S. pombe, the humanCBF2 andc-myc genes, and the kanR gene as heterologous genes and markers. Printing buffer was deposited on the empty spots. The array shown results from hybridization with cDNA targets of total RNA isolated from wild-type BY4742 (Cy3-labelled) and Δydr225w (Cy5-labelled) strain.
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Related In: Results  -  Collection

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Figure 6: A view of the microarray. The array is composed of 32 grids of 420 spots each. A total of ca. 200 empty spots are distributed through the array for background controls. Probe spots are deposited in close duplicates. A set of PCR products and synthetic oligonucleotides was selected as controls. These include scorecard kits (Amersham Biosciences) in grids 4, 8, 12, 16, 20, 24, 28, and 32; serial dilutions of the signal normalization luciferase gene (row 1 in grids 3, 7, 11, 15, 19, 23, 27, 31) for which control RNA spike (Promega) can be obtained; 10 long oligonucleotides covering YKL182w (6153 bp) and 9 long oligonucleotides covering YLR310c (4767 bp) ORFs as controls for reverse transcription efficiency; 10 intergenic regions, 10 intronic sequences, and mitochondrial genes, 20 non-monotonous trinucleotide repeats (72-mer oligonucleotides) and 4 serial dilutions (in grid 1, 4, 29 and 32) of the total genomic DNA from the wild-type strain S288c; 3 E. coli genes (tufA, aceF, kdtA) as negative controls; the LexA binding domain, the LacZ 5' and 3'end regions, the Pho4 binding domain, the Gal4 binding domain, the GFP, the TAP and GST as commonly used tags or reporter genes; Leu1, His5, and Ura4 from S. pombe, the humanCBF2 andc-myc genes, and the kanR gene as heterologous genes and markers. Printing buffer was deposited on the empty spots. The array shown results from hybridization with cDNA targets of total RNA isolated from wild-type BY4742 (Cy3-labelled) and Δydr225w (Cy5-labelled) strain.
Mentions: Using the robot SDDC-2 (Engineering Services Inc., ESI), DNAs (PCR products, 71-mer oligonucleotides, and control genes) were deposited in close duplicates (1 fmol DNA of PCR product and 10 fmol DNA of long oligonucleotide) onto microarray aldehyde glass slides with 200-μm spacing between neighbouring spot centers. Subsequent microarray treatments were performed as recommended by the manufacturer (Telechem International). The microarray view is described in Figure 6. Note that batches 800, 400, and 616 (prepared successively in this order from November 2001 to February 2002) used in our hybridization experiments differ from the final ones described above, since the probes corresponding to the genes YBR145w, YDR225w, YEL033w, YGL147c, YGR148c, YJR148w, YMR011w, YMR169c, YMR170c, and YNL143c are present in the form of PCR products instead of 71-mer oligonucleotides.

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