On-chip non-equilibrium dissociation curves and dissociation rate constants as methods to assess specificity of oligonucleotide probes

Abstract

Nucleic acid hybridization serves as backbone for many high-throughput systems for detection, expression analysis, comparative genomics and re-sequencing. Specificity of hybridization between probes and intended targets is always critical. Approaches to ensure and evaluate specificity include use of mismatch probes, obtaining dissociation curves rather than single temperature hybridizations, and comparative hybridizations. In this study, we quantify effects of mismatch type and position on intensity of hybridization signals and provide a new approach based on dissociation rate constants to evaluate specificity of hybridized signals in complex target mixtures. Using an extensive set of 18mer oligonucleotide probes on an in situ synthesized biochip platform, we demonstrate that mismatches in the center of the probe are more discriminating than mismatches toward the extremities of the probe and mismatches toward the attached end are less discriminating than those toward the loose end. The observed destabilizing effect of a mismatch type agreed in general with predictions using the nearest neighbor model. Use of a new parameter, specific dissociation temperature (T(d-w), temperature of maximum specific dissociation rate constant), obtained from probe-target duplex dissociation profiles considerably improved the evaluation of specificity. These results have broad implications for hybridization data obtained from complex mixtures of nucleic acids.

Figure 1

Dissociation profiles from a perfect match (filled triangles) probe and two probes containing a single base pair mismatch (empty and filled circles). (A) Normalized signal intensities. Probe sequences are given with the mismatches in bold italics. T_d-50 values are indicated by arrows. (B) Dissociation rate constants. Dashed lines show the best fit of values measured between 30 and 1°C below the maximum k_d to the Arrhenius equation. T_d-w values are indicated by arrows.

Figure 2

Correlation between T_m and T_d-w (A), and T_m and T_d-50 (B) for PM probes. Black circles: 18mer, not saturated (n = 819); open triangles: 18 and 20mer saturated (n = 25). Values are means of five experiments. The R² value for the linear fit between T_m and T_d is given for not saturated 18mer.

Figure 3

Influence of the position of a single base pair mismatch on the initial signal intensity (A) and on ΔT_d-w (B). Boxes indicate the range from the 25th to 75th percentile, whiskers the 10th and the 90th percentile. The median is given as a solid, the mean as a dotted line. Sample sizes are given for each position (n =). In (A) the percentage of MM probes with signal intensities below 5 SD of background are given (%). In (B) ‘%’ shows what percentage of all probes with the MM at that position had good quality dissociation curves and was used for analysis. Note that for positions 5–11, <40% of probes gave good quality measurement of T_d-w. Values in this range are likely an underestimation. Probes are attached to the chip at the 3′ end (position 18).

Figure 4

Designation of MM-types. In naming MM-types, the first letter denotes the base in the probe (solid line), the second the one in the target (dashed line). An example for a T-C (middle) and a C-T (right) mismatch with the corresponding PM probe (left) are given.

Figure 5

Correlation between predicted and measured effects of mismatch type on initial signal intensity (A), and on ΔT_d-w (B). Group means with 95% confidence intervals are given. R² values are for the best linear fit between calculated and measured group means. See Table 1 for sample sizes.

Figure 6

Normalized dissociation curves (top panels A, C, E) and dissociation rate constants k_d (bottom panels B, D, F) of three PM probes showing various degrees of cross-hybridization. Hybridization was carried out with 4.2 µg of PCR-products only (filled circles), 3.4 µg genomic DNA spiked with 400 ng PCR products (empty circles), 3.7 µg genomic DNA spiked with 45 ng PCR products (filled triangles) and 3.8 µg genomic DNA only (empty triangles). Gene targets and probe sequences are given in the upper panels.

Figure 7

Impact of cross-hybridization on computed T_d-w (A and B) or T_d-50 (C and D). For cross-hybridization, 3.4 µg genomic DNA was spiked with 400 ng PCR products (A and C) or 3.7 µg genomic DNA was spiked with 45 ng PCR products (B and D). Both were compared with T_d-w or T_d-50 obtained using PCR products only (n = 704). Values for PM probes where the signal intensity of an MM probe (with MM at position 9) was higher are shown as open triangles.