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Oliz, a suite of Perl scripts that assist in the design of microarrays using 50mer oligonucleotides from the 3' untranslated region.

Chen H, Sharp BM - BMC Bioinformatics (2002)

Bottom Line: These sequences were analyzed for gene specificity.Five Cy3-labeled cDNAs were used to empirically verify the specificity of a set of 1814 50mers.Oliz can be used to select oligonucleotide sequences for microarrays.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Tennessee Health Science Center, Memphis, TN 38163, USA. hchen@utmem.edu

ABSTRACT

Background: Identifying reliable oligonucleotide sequences for use in microarray experiments is a complex process. Two key issues are the accuracy of the input sequences and the specificity of the oligonucleotide sequences.

Results: We provide a suite of Perl scripts that facilitates the search for gene-specific oligonucleotides for microarray experiments. Genes of interest are first identified in the form of UniGene clusters. The sequences of these clusters were extracted and assembled into contigs to increase their accuracy. The 3' untranslated region (3'UTR) of the contig was parsed. Then, multiple 50mer oligonucleotide sequences with similar melting temperature were obtained from each 3'UTR. These sequences were analyzed for gene specificity. Five Cy3-labeled cDNAs were used to empirically verify the specificity of a set of 1814 50mers.

Conclusion: Oliz can be used to select oligonucleotide sequences for microarrays. Oliz is freely available for academic users at http://www.utmem.edu/pharmacology/otherlinks/oliz.html

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Related in: MedlinePlus

Verification of specificity. Microarrays were printed with 1816 oligonucleotides in duplicate. Each sub-grid has 13 columns and 8 rows. (A) SYTO59 DNA staining demonstrating the layout of one sub-grid. The spot in the upper right corner and the spot four columns to the left were printed with Cy3-labeled oligonucleotides for orientation purposes. Some positions near the top of each sub-grid were printed with buffer only, and are thus not visible on SYTO59 staining. (B)-(F): cDNAs contain the complementary strand of one of the 50mers on the array were amplified by PCR and labeled with Cy3. Hybridization of these cDNAs to the microarray shows that only the complementary spot had fluorescent signal in most cases. cDNA for Rn.2401 also hybridized to another spot in the same sub-grid, but with lower intensity.
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Figure 2: Verification of specificity. Microarrays were printed with 1816 oligonucleotides in duplicate. Each sub-grid has 13 columns and 8 rows. (A) SYTO59 DNA staining demonstrating the layout of one sub-grid. The spot in the upper right corner and the spot four columns to the left were printed with Cy3-labeled oligonucleotides for orientation purposes. Some positions near the top of each sub-grid were printed with buffer only, and are thus not visible on SYTO59 staining. (B)-(F): cDNAs contain the complementary strand of one of the 50mers on the array were amplified by PCR and labeled with Cy3. Hybridization of these cDNAs to the microarray shows that only the complementary spot had fluorescent signal in most cases. cDNA for Rn.2401 also hybridized to another spot in the same sub-grid, but with lower intensity.

Mentions: All of the five Cy3-labeled cDNAs hybridized to their expected spots. The subgrids (13 × 8 spots) that contain the specific hybridized spots are shown in Figure 2. Two spots on the array, known to have green autofluorescence (not shown), were excluded from the analysis. Depending on the specific cDNA sequence, there were 0–4 additional spots that had detectable fluorescence. This represents 0–4/1814 × 100% = 0–0.22% of all spots. No non-specific spots was associated with more than one cDNA. The intensity of these spots are shown in Table 1. Since the duplicates within each microarray slide had similar intensity values, the average intensity is given. The level of non-specific binding ranged from approximately 5–15% of specific binding; only 25% of the non-specific spots had fluorescence binding values that were > 10% of specific binding. The magnitude of non-specific binding was not related to the level of specific binding.


Oliz, a suite of Perl scripts that assist in the design of microarrays using 50mer oligonucleotides from the 3' untranslated region.

Chen H, Sharp BM - BMC Bioinformatics (2002)

Verification of specificity. Microarrays were printed with 1816 oligonucleotides in duplicate. Each sub-grid has 13 columns and 8 rows. (A) SYTO59 DNA staining demonstrating the layout of one sub-grid. The spot in the upper right corner and the spot four columns to the left were printed with Cy3-labeled oligonucleotides for orientation purposes. Some positions near the top of each sub-grid were printed with buffer only, and are thus not visible on SYTO59 staining. (B)-(F): cDNAs contain the complementary strand of one of the 50mers on the array were amplified by PCR and labeled with Cy3. Hybridization of these cDNAs to the microarray shows that only the complementary spot had fluorescent signal in most cases. cDNA for Rn.2401 also hybridized to another spot in the same sub-grid, but with lower intensity.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC130177&req=5

Figure 2: Verification of specificity. Microarrays were printed with 1816 oligonucleotides in duplicate. Each sub-grid has 13 columns and 8 rows. (A) SYTO59 DNA staining demonstrating the layout of one sub-grid. The spot in the upper right corner and the spot four columns to the left were printed with Cy3-labeled oligonucleotides for orientation purposes. Some positions near the top of each sub-grid were printed with buffer only, and are thus not visible on SYTO59 staining. (B)-(F): cDNAs contain the complementary strand of one of the 50mers on the array were amplified by PCR and labeled with Cy3. Hybridization of these cDNAs to the microarray shows that only the complementary spot had fluorescent signal in most cases. cDNA for Rn.2401 also hybridized to another spot in the same sub-grid, but with lower intensity.
Mentions: All of the five Cy3-labeled cDNAs hybridized to their expected spots. The subgrids (13 × 8 spots) that contain the specific hybridized spots are shown in Figure 2. Two spots on the array, known to have green autofluorescence (not shown), were excluded from the analysis. Depending on the specific cDNA sequence, there were 0–4 additional spots that had detectable fluorescence. This represents 0–4/1814 × 100% = 0–0.22% of all spots. No non-specific spots was associated with more than one cDNA. The intensity of these spots are shown in Table 1. Since the duplicates within each microarray slide had similar intensity values, the average intensity is given. The level of non-specific binding ranged from approximately 5–15% of specific binding; only 25% of the non-specific spots had fluorescence binding values that were > 10% of specific binding. The magnitude of non-specific binding was not related to the level of specific binding.

Bottom Line: These sequences were analyzed for gene specificity.Five Cy3-labeled cDNAs were used to empirically verify the specificity of a set of 1814 50mers.Oliz can be used to select oligonucleotide sequences for microarrays.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pharmacology, University of Tennessee Health Science Center, Memphis, TN 38163, USA. hchen@utmem.edu

ABSTRACT

Background: Identifying reliable oligonucleotide sequences for use in microarray experiments is a complex process. Two key issues are the accuracy of the input sequences and the specificity of the oligonucleotide sequences.

Results: We provide a suite of Perl scripts that facilitates the search for gene-specific oligonucleotides for microarray experiments. Genes of interest are first identified in the form of UniGene clusters. The sequences of these clusters were extracted and assembled into contigs to increase their accuracy. The 3' untranslated region (3'UTR) of the contig was parsed. Then, multiple 50mer oligonucleotide sequences with similar melting temperature were obtained from each 3'UTR. These sequences were analyzed for gene specificity. Five Cy3-labeled cDNAs were used to empirically verify the specificity of a set of 1814 50mers.

Conclusion: Oliz can be used to select oligonucleotide sequences for microarrays. Oliz is freely available for academic users at http://www.utmem.edu/pharmacology/otherlinks/oliz.html

Show MeSH
Related in: MedlinePlus