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

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.

Keywords: cross-hybridization; hybridization; microarray; probe length; probe-level signal.

Figure 1

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.

Figure 2

Effects of variation of hybridization, wash, and probe conditions on signal intensities identified by titration assay of hybridization with target oligonucleotide, Dap3-02, without cDNA background. The line plots show absolute signal intensities of PM and MM probes. The bar graphs show ratios of signal intensity of PM to that of cognate MM. The average signal intensities determined using GCOS 1.0 software were derived from two replicate GeneChip analyses except for the experiment regarding wash conditions. The signal intensities under stringent wash conditions were derived from a single GeneChip analysis. (A) Hybridization temperature. (B) Duration of hybridization. (C) Number of stringent wash cycles. (D) Setting of adjacent probe pairs of AFFX-DapX-3_at No. 02, such as AFFX-DapX-3_at No. 01, No. 03, and No. 04, and the possible duplexes formed between target oligonucleotide, Dap3-02, and the adjacent probes. The constitutive part of the sense strand of the spiked probe, Dap, is shown as the target sequence. The middle four sequences are adjacent probe pairs and the flip-flop position is underlined. The possible base pairs hybridizing with target oligonucleotide, Dap3-02, are indicated by asterisks and the numbers beside the sequences indicate the numbers of hybridizing base pairs. The sequence of the target oligonucleotide, Dap3-02, is shown at the bottom. The gray nucleotides in the 3′ end of Dap3-02 indicate the excised six bases in the target oligonucleotide, Dap3-02-6nt, from which results are shown in Figures 3 and 4B. (E) Absolute signal intensities of adjacent probe pairs, AFFX-DapX-3_at No. 01, No. 03, and No. 04.

Figure 3

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.

Figure 4

Absolute signal intensities of adjacent probe pairs, AFFX-DapX-3_at No. 01, No. 03, and No. 04 with cDNA background. (A) PM, MM signals, and PM/MM ratios, which were demonstrated by the titration assay of hybridization with target oligonucleotide Dap3-02, with cDNA background. 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. (B) Effects of shortening target length on absolute signal intensities with 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 bar graphs show the ratios of signal intensity of PM to those of cognate MM probes. The average signal intensities were determined as described above.