Limits...
Insight into the sequence specificity of a probe on an Affymetrix GeneChip by titration experiments using only one oligonucleotide

View Article: PubMed Central - PubMed

ABSTRACT

High-density oligonucleotide arrays are powerful tools for the analysis of genome-wide expression of genes and for genome-wide screens of genetic variation in living organisms. One of the critical problems in high-density oligonucleotide arrays is how to identify the actual amounts of a transcript due to noise and cross-hybridization involved in the observed signal intensities. Although mismatch (MM) probes are spotted on Affymetrix GeneChips to evaluate the noise and cross-hybridization embedded in perfect match (PM) probes, the behavior of probe-level signal intensities remains unclear. In the present study, we hybridized only one complement 25-mer oligonucleotide to characterize the behavior of duplex formation between target and probe in the complete absence of cross-hybridization. Titration experiments using only one oligonucleotide demonstrated that a substantial amount of intact target was hybridized not only to the PM but also the MM probe and that duplex formation between intact target and MM probe was efficiently reduced by increasing the stringency of hybridization conditions and shortening probe length. In addition, we discuss the correlation between potential for secondary structure of target oligonucleotide and hybridization intensity. These findings will be useful for the development of genome-wide analysis of gene expression and genetic variations by optimization of hybridization and probe conditions.

No MeSH data available.


Effects of shortening target length on absolute signal intensities without cDNA background. The line plots show absolute signal intensities of PM and MM probes, which were increased by titration assay of hybridization with target oligonucleotide, Dap3-02-6nt. The line plots show absolute signal intensities of PM and MM probes. The bar graphs show ratios of signal intensity of PM to those of cognate MM probes. The average signal intensities determined using GCOS 1.0 software were derived from two replicate GeneChip analyses.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC5036658&req=5

f3-3_47: Effects of shortening target length on absolute signal intensities without cDNA background. The line plots show absolute signal intensities of PM and MM probes, which were increased by titration assay of hybridization with target oligonucleotide, Dap3-02-6nt. The line plots show absolute signal intensities of PM and MM probes. The bar graphs show ratios of signal intensity of PM to those of cognate MM probes. The average signal intensities determined using GCOS 1.0 software were derived from two replicate GeneChip analyses.

Mentions: Next, to confirm that the number of base pairs involved in duplex formation was important for increasing the PM/MM ratio, the 19-mer oligonucleotide target, Dap3-02-6nt, which was six bases shorter than Dap3-02 from the 3′ end, was hybridized to the GeneChip Test3 Array at 45°C for 16 h. Figure 3 shows the absolute signal intensities of adjacent probe pairs, AFFX-DapX-3_at No. 01, No. 03, and No. 04, that were increased by the titration assay of hybridization with 19-mer Dap3-02-6nt target oligonucleotide. The bar graph shows increases in PM/MM ratios in AFFX-DapX-3_at No. 01 and No. 02 probe pairs. Although the number of base pairs involved in duplex formation decreased, the PM signal intensities of AFFX-DapX-3_at No. 02 increased in the target concentration range of 14 fM to 1.4 pM, in contrast to our expectations.


Insight into the sequence specificity of a probe on an Affymetrix GeneChip by titration experiments using only one oligonucleotide
Effects of shortening target length on absolute signal intensities without cDNA background. The line plots show absolute signal intensities of PM and MM probes, which were increased by titration assay of hybridization with target oligonucleotide, Dap3-02-6nt. The line plots show absolute signal intensities of PM and MM probes. The bar graphs show ratios of signal intensity of PM to those of cognate MM probes. The average signal intensities determined using GCOS 1.0 software were derived from two replicate GeneChip analyses.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036658&req=5

f3-3_47: Effects of shortening target length on absolute signal intensities without cDNA background. The line plots show absolute signal intensities of PM and MM probes, which were increased by titration assay of hybridization with target oligonucleotide, Dap3-02-6nt. The line plots show absolute signal intensities of PM and MM probes. The bar graphs show ratios of signal intensity of PM to those of cognate MM probes. The average signal intensities determined using GCOS 1.0 software were derived from two replicate GeneChip analyses.
Mentions: Next, to confirm that the number of base pairs involved in duplex formation was important for increasing the PM/MM ratio, the 19-mer oligonucleotide target, Dap3-02-6nt, which was six bases shorter than Dap3-02 from the 3′ end, was hybridized to the GeneChip Test3 Array at 45°C for 16 h. Figure 3 shows the absolute signal intensities of adjacent probe pairs, AFFX-DapX-3_at No. 01, No. 03, and No. 04, that were increased by the titration assay of hybridization with 19-mer Dap3-02-6nt target oligonucleotide. The bar graph shows increases in PM/MM ratios in AFFX-DapX-3_at No. 01 and No. 02 probe pairs. Although the number of base pairs involved in duplex formation decreased, the PM signal intensities of AFFX-DapX-3_at No. 02 increased in the target concentration range of 14 fM to 1.4 pM, in contrast to our expectations.

View Article: PubMed Central - PubMed

ABSTRACT

High-density oligonucleotide arrays are powerful tools for the analysis of genome-wide expression of genes and for genome-wide screens of genetic variation in living organisms. One of the critical problems in high-density oligonucleotide arrays is how to identify the actual amounts of a transcript due to noise and cross-hybridization involved in the observed signal intensities. Although mismatch (MM) probes are spotted on Affymetrix GeneChips to evaluate the noise and cross-hybridization embedded in perfect match (PM) probes, the behavior of probe-level signal intensities remains unclear. In the present study, we hybridized only one complement 25-mer oligonucleotide to characterize the behavior of duplex formation between target and probe in the complete absence of cross-hybridization. Titration experiments using only one oligonucleotide demonstrated that a substantial amount of intact target was hybridized not only to the PM but also the MM probe and that duplex formation between intact target and MM probe was efficiently reduced by increasing the stringency of hybridization conditions and shortening probe length. In addition, we discuss the correlation between potential for secondary structure of target oligonucleotide and hybridization intensity. These findings will be useful for the development of genome-wide analysis of gene expression and genetic variations by optimization of hybridization and probe conditions.

No MeSH data available.