J. Phys. Chem. B 2007, 111, 13583-13590
13583
Thermodynamic Behavior of Short Oligonucleotides in Microarray Hybridizations Can Be Described Using Gibbs Free Energy in a Nearest-Neighbor Model Stefan Weckx,†,‡ Enrico Carlon,§,|,⊥ Luc De Vuyst,‡ and Paul Van Hummelen*,†,# MicroArray Facility, VIB, Herestraat 49, B-3000 LeuVen, Belgium, Interdisciplinary Research Institute, Cite´ Scientifique BP 60069, F-59652 VilleneuVe d’Ascq, France, Ecole Polytechnique UniVersitaire de Lille, Cite´ Scientifique, F-59655 VilleneuVe d’Ascq, France, Institute for Theoretical Physics, K. U. LeuVen, Celestijnenlaan 200D, B-3001 LeuVen, Belgium, Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Vrije UniVersiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium, and MicroArray Facility, K. U. LeuVen, Herestraat 49, B-3000 LeuVen, Belgium ReceiVed: July 4, 2007; In Final Form: September 21, 2007
While designing oligonucleotide-based microarrays, cross-hybridization between surface-bound oligos and non-intended labeled targets is probably the most difficult parameter to predict. Although literature describes rules-of-thumb concerning oligo length, overall similarity, and continuous stretches, the final behavior is difficult to predict. The aim of this study was to investigate the effect of well-defined mismatches on hybridization specificity using CodeLink Activated Slides and to study quantitatively the relation between hybridization intensity and Gibbs free energy (∆G), taking the mismatches into account. Our data clearly showed a correlation between the hybridization intensity and ∆G of the oligos over 3 orders of magnitude for the hybridization intensity, which could be described by the Langmuir model. As ∆G was calculated according to the nearest-neighbor model, using values related to DNA hybridizations in solution, this study clearly shows that target-probe hybridizations on microarrays with a three-dimensional coating are in quantitative agreement with the corresponding reaction in solution. These results can be interesting for some practical applications. The correlation between intensity and ∆G can be used in quality control of microarray hybridizations by designing probes and corresponding RNA spikes with a range of ∆G values. Furthermore, this correlation might be of use to fine-tune oligonucleotide design algorithms in a way to improve the prediction of the influence of mismatching targets on microarray hybridizations.
Introduction In transcriptome research, microarrays are used to indicate genes expressed by a given cell type or organism. The overall expression pattern is in most cases compared to other patterns to reveal differentially expressed genes between different states or experimental conditions. Microarray technology is based on nucleotide-nucleotide hybridizations, where one molecule (the probe) is attached to the surface of a slide, and the other molecule (the fluorescently labeled target) moves freely in solution and will bind to its complementary probe. To obtain a microarray with high sensitivity and specificity, the design of probe sequences is an important step. A good sensitivity results in intensities proportional to the real amount of labeled target in the hybridization solution. To obtain a good specificity, homology should be high for the intended target sequence but low for all other target sequences in solution to avoid crosshybridization of non-intended similar sequences. Previous work described in the literature presents hybridization studies with respect to sensitivity, specificity, crosshybridization, mismatches, oligo length, and number of oligos * Corresponding author. Tel.: +32 16 347939; fax: +32 16 347940; e-mail:
[email protected]. † MicroArray Facility, VIB. ‡ Research Group of Industrial Microbiology and Food Biotechnology. § Interdisciplinary Research Institute c/o IEMN. | Universitaire de Lille. ⊥ Institute for Theoretical Physics. # MicroArray Facility, K. U. Leuven.
per gene.1-5 Kane et al.1 demonstrated that 50-mer oligonucleotides have a comparable sensitivity as PCR-derived probes of 300-400 nucleotides and that 50-mers show good specificity as long as the similarity with non-intended sequences is
