Hybridization Isotherms of DNA Microarrays and the Quantification of Mutation Studies

Abstract

Background: Diagnostic DNA arrays for detection of point mutations as markers for cancer usually function in the presence of a large excess of wild-type DNA. This excess can give rise to false positives as a result of competitive hybridization of the wild-type target at the mutation spot. Analysis of the DNA array data is typically qualitative, aimed at establishing the presence or absence of a particular point mutation. Our theoretical approach yields methods for quantifying the analysis to obtain the ratio of concentrations of mutated and wildtype DNA. Method: The theory is formulated in terms of the hybridization isotherms relating the hybridization fraction at the spot to the composition of the sample solutions at thermodynamic equilibrium. It focuses on samples containing an excess of single-stranded DNA and on DNA arrays with a low surface density of probes. The hybridization equilibrium constants can be obtained by the nearest-neighbor method. Results: Two approaches allow acquisition of quantitative results from the DNA array data. In one, the signal of the mutation spot is compared with that of the wild-type spot. The implementation requires knowledge of the saturation intensity of the two spots. The second approach requires comparison of the intensity of the mutation spot at two different temperatures. In this case, knowledge of the saturation signal is not always necessary. Conclusions: DNA arrays can be used to obtain quantitative results on the concentration ratio of mutated

Thermostable DNA Immobilization and Temperature Effects on Surface Hybridization

“…Equations 2 can be combined and rearranged to yield an expression for the equilibrium fraction f FM of probes that hybridize to fully matched targets 51–52 …”

Salt Concentration Effects on Equilibrium Melting Curves from DNA Microarrays

“…Due to the phosphate charges along the DNA backbone, the effect of salt concentration and the electrostatic penalty for hybridization was considered (12,13). Furthermore, competitive hybridization on the surface and/or in solution is present due to the mixture of different targets affecting the hybridization kinetics and the available target concentrations (7,(14)(15)(16)(17). To test the different models, dedicated experiments are necessary for which the fabrication parameters are controlled and the target solutions well known (2).…”

Brush Effects on DNA Chips: Thermodynamics, Kinetics, and Design Guidelines

“…A growing theory effort aims to clarify the underlying physics of DNA chips with view of assisting in their design and in the analysis of the results. The Langmuir isotherm and the corresponding kinetic scheme provide a natural starting point for the modeling (Chan et al, 1995;Livshits and Mirzabekov, 1996;Vainrub and Pettitt, 2002;Bhanot et al, 2003;Held et al, 2003;Zhang et al, 2003;Halperin et al, 2004aHalperin et al, , 2004b as well as the analysis of the experimental results (Forman et al, 1998;Okahata et al, 1998;Steel et al, 1998;Georgiadis et al, 2000;Nelson et al, 2001;Dai et al, 2002;Kepler et al, 2002;Peterson et al, 2002;Hekstra et al, 2003). Within this model, the probes, irrespective of their hybridization state, do not interact.…”