Clinical Chemistry
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Clinical Chemistry 50: 2254-2262, 2004. First published September 23, 2004; 10.1373/clinchem.2004.037226
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(Clinical Chemistry. 2004;50:2254-2262.)
© 2004 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Hybridization Isotherms of DNA Microarrays and the Quantification of Mutation Studies

Avraham Halperin1,a, Arnaud Buhot1 and Ekaterina B. Zhulina2

1 Unité Mixte de Recherche (UMR) 5819 [Université Joseph Fourier (UJF), Centre National de la Recherche Scientifique (CNRS), Commissariat à l’Energie Atomique (CEA)], DRFMC/SI3M, CEA Grenoble, Grenoble, France.
2 Institute of Macromolecular Compounds of the Russian Academy of Sciences, Bolshoy, Russia.

aAddress correspondence to this author at: UMR 5819 (UJF, CNRS, CEA), DRFMC/SI3M, CEA Grenoble, 17 rue des Martyrs, 38054, Grenoble cedex 9, France. Fax 33-4-3878-5691; e-mail ahalperin{at}cea.fr.

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 wild-type 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 DNA to wild-type DNA in studies of somatic point mutations.






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