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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.


Absolute signal intensities of perfect match (PM) and mismatch (MM) probes, such as AFFX-DapX-5_at No. 11, AFFX-DapX-M_at No. 19, and AFFX-DapX-3_at No. 02, which were identified by independent titration assay of hybridization with DNA target oligos, Dap5-11, DapM-19, and Dap3-02, respectively. Bar graphs show the 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. (A) PM, MM signals, and PM/MM ratios with cDNA background. (B) PM, MM signals, and PM/MM ratios without cDNA background.
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f1-3_47: Absolute signal intensities of perfect match (PM) and mismatch (MM) probes, such as AFFX-DapX-5_at No. 11, AFFX-DapX-M_at No. 19, and AFFX-DapX-3_at No. 02, which were identified by independent titration assay of hybridization with DNA target oligos, Dap5-11, DapM-19, and Dap3-02, respectively. Bar graphs show the 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. (A) PM, MM signals, and PM/MM ratios with cDNA background. (B) PM, MM signals, and PM/MM ratios without cDNA background.

Mentions: To characterize the absolute signal intensities of perfect match (PM) and mismatch (MM) probes precisely, target oligonucleotides labeled at the 3′ end with biotin were hybridized to the GeneChip Test3 Array (Affymetrix) with and without cDNA background generated from Escherichia coli total RNA under standard hybridization conditions. We chose three target oligonucleotides, Dap5-11, DapM-19, and Dap3-02, which were complementary to the spiked control probes, AFFX-DapX-5_at No. 11, AFFX-DapX-M_at No. 19, and AFFX-DapX-3_at No. 02, respectively. These target oligonucleotides had the same GC content of 56% to exclude the effect of GC content-dependent hybridization strength. Recently, hybridization models based on the nearest neighbor model18 were reported10,12,13, and we predicted Gibbs free energy of target oligonucleotides. The nearest neighbor model predicted that the potentials for duplex formation, ΔG, of oligonucleotide targets, Dap5-11, DapM-19, and Dap3-02, were −31.3, −29.7, and −30.0 kcal/mmol, respectively. Figures 1A and 1B show the signal intensities and ratios of signal intensity of PM to that of cognate MM of AFFX-DapX-5_at No. 11, AFFX-DapX-M_at No. 19, and AFFX-DapX-3_at No. 02 probe pairs as a function of target oligonucleotide concentration with and without cDNA background. Although all three target oligonucleotides with cDNA background were applied to the Test3 Array at once, only one target oligonucleotide was hybridized to the array without cDNA background to avoid the effects of cross-hybridization completely. Both with and without cDNA background, the line plots of intensity vs. target concentration showed the typical sigmoidal shape encountered in chemical kinetics and the signal intensity was saturated at a target concentration of 140 pM. However, the detection limit depended heavily on the abundance of the background cDNA. Although the detection limit was at a concentration of ca. 1.4 fM without background cDNA, addition of background cDNA caused a shift in the detection limit to ca. 1.4 pM. It follows that linearity was changed from the target oligonucleotide concentration range of 14 fM–14 pM to 1.4 pM–14 pM due to addition of cDNA background, depending on the variety of the target sequences. These results indicated that cDNA background conceals the behavior of target-probe hybridization at less than or equal to a target concentration of 140 fM. Particularly in the case of Dap3-02, substantial signal intensities derived from background cDNA were observed, although the three target oligonucleotides used in the present study had the same GC content of 56% and similar potentials for duplex formation. Moreover, in the case of Dap5-11, signal intensities of MM probes were stronger than those of PM probes in the target oligonucleotide concentration range of 1.4 fM–140 fM. Our findings further supported the importance of evaluation of the background signal embedded in PM probes. Consequently, we focused on analysis without cDNA background to characterize the hybridization behavior of PM and MM probes in the low concentrations of target oligonucleotides. It is worth noting that signal intensities of MM probes increased with increasing target concentration despite the complete absence of cross-hybridization (Fig. 1B). This result clearly indicated that a substantial amount of intact target was hybridized not only to the PM but also to the MM probe, even if the target oligonucleotides were applied at low concentrations. In Figure 1B, the horizontal position of the curves reflects differences in target-binding strength for hybridization among them, although the target oligonucleotides had the same GC content and similar potentials for duplex formation. Although there was a small difference in the potentials for duplex formation of oligonucleotide targets, the order of predicted free energy does not seem to reflect the observed signal intensity. Possible reason for this discrepancy is addressed in Discussion. The bar graph shows the ratios of signal intensities of PM to those of cognate MM probes. The PM/MM ratio is an index of specificity and is especially important in re-sequencing experiments in screening of genetic variation. Although the ratios of signal intensity of PM to that of cognate MM were almost 2, there was little variation. It is likely that the largest PM/MM ratio was correlated with target-binding strength. Briefly, the PM/MM ratio of DapM-19, which shows high target-binding strength, was the largest at the comparatively low target concentration of 140 fM, while that of Dap5-11, which shows low target-binding strength, was largest at the high target concentration at 14 pM.


Insight into the sequence specificity of a probe on an Affymetrix GeneChip by titration experiments using only one oligonucleotide
Absolute signal intensities of perfect match (PM) and mismatch (MM) probes, such as AFFX-DapX-5_at No. 11, AFFX-DapX-M_at No. 19, and AFFX-DapX-3_at No. 02, which were identified by independent titration assay of hybridization with DNA target oligos, Dap5-11, DapM-19, and Dap3-02, respectively. Bar graphs show the 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. (A) PM, MM signals, and PM/MM ratios with cDNA background. (B) PM, MM signals, and PM/MM ratios without cDNA background.
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Related In: Results  -  Collection

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f1-3_47: Absolute signal intensities of perfect match (PM) and mismatch (MM) probes, such as AFFX-DapX-5_at No. 11, AFFX-DapX-M_at No. 19, and AFFX-DapX-3_at No. 02, which were identified by independent titration assay of hybridization with DNA target oligos, Dap5-11, DapM-19, and Dap3-02, respectively. Bar graphs show the 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. (A) PM, MM signals, and PM/MM ratios with cDNA background. (B) PM, MM signals, and PM/MM ratios without cDNA background.
Mentions: To characterize the absolute signal intensities of perfect match (PM) and mismatch (MM) probes precisely, target oligonucleotides labeled at the 3′ end with biotin were hybridized to the GeneChip Test3 Array (Affymetrix) with and without cDNA background generated from Escherichia coli total RNA under standard hybridization conditions. We chose three target oligonucleotides, Dap5-11, DapM-19, and Dap3-02, which were complementary to the spiked control probes, AFFX-DapX-5_at No. 11, AFFX-DapX-M_at No. 19, and AFFX-DapX-3_at No. 02, respectively. These target oligonucleotides had the same GC content of 56% to exclude the effect of GC content-dependent hybridization strength. Recently, hybridization models based on the nearest neighbor model18 were reported10,12,13, and we predicted Gibbs free energy of target oligonucleotides. The nearest neighbor model predicted that the potentials for duplex formation, ΔG, of oligonucleotide targets, Dap5-11, DapM-19, and Dap3-02, were −31.3, −29.7, and −30.0 kcal/mmol, respectively. Figures 1A and 1B show the signal intensities and ratios of signal intensity of PM to that of cognate MM of AFFX-DapX-5_at No. 11, AFFX-DapX-M_at No. 19, and AFFX-DapX-3_at No. 02 probe pairs as a function of target oligonucleotide concentration with and without cDNA background. Although all three target oligonucleotides with cDNA background were applied to the Test3 Array at once, only one target oligonucleotide was hybridized to the array without cDNA background to avoid the effects of cross-hybridization completely. Both with and without cDNA background, the line plots of intensity vs. target concentration showed the typical sigmoidal shape encountered in chemical kinetics and the signal intensity was saturated at a target concentration of 140 pM. However, the detection limit depended heavily on the abundance of the background cDNA. Although the detection limit was at a concentration of ca. 1.4 fM without background cDNA, addition of background cDNA caused a shift in the detection limit to ca. 1.4 pM. It follows that linearity was changed from the target oligonucleotide concentration range of 14 fM–14 pM to 1.4 pM–14 pM due to addition of cDNA background, depending on the variety of the target sequences. These results indicated that cDNA background conceals the behavior of target-probe hybridization at less than or equal to a target concentration of 140 fM. Particularly in the case of Dap3-02, substantial signal intensities derived from background cDNA were observed, although the three target oligonucleotides used in the present study had the same GC content of 56% and similar potentials for duplex formation. Moreover, in the case of Dap5-11, signal intensities of MM probes were stronger than those of PM probes in the target oligonucleotide concentration range of 1.4 fM–140 fM. Our findings further supported the importance of evaluation of the background signal embedded in PM probes. Consequently, we focused on analysis without cDNA background to characterize the hybridization behavior of PM and MM probes in the low concentrations of target oligonucleotides. It is worth noting that signal intensities of MM probes increased with increasing target concentration despite the complete absence of cross-hybridization (Fig. 1B). This result clearly indicated that a substantial amount of intact target was hybridized not only to the PM but also to the MM probe, even if the target oligonucleotides were applied at low concentrations. In Figure 1B, the horizontal position of the curves reflects differences in target-binding strength for hybridization among them, although the target oligonucleotides had the same GC content and similar potentials for duplex formation. Although there was a small difference in the potentials for duplex formation of oligonucleotide targets, the order of predicted free energy does not seem to reflect the observed signal intensity. Possible reason for this discrepancy is addressed in Discussion. The bar graph shows the ratios of signal intensities of PM to those of cognate MM probes. The PM/MM ratio is an index of specificity and is especially important in re-sequencing experiments in screening of genetic variation. Although the ratios of signal intensity of PM to that of cognate MM were almost 2, there was little variation. It is likely that the largest PM/MM ratio was correlated with target-binding strength. Briefly, the PM/MM ratio of DapM-19, which shows high target-binding strength, was the largest at the comparatively low target concentration of 140 fM, while that of Dap5-11, which shows low target-binding strength, was largest at the high target concentration at 14 pM.

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.