Parallel thermodynamic analysis of duplexes on oligodeoxyribonucleotide microchips

Abstract

A microchip method has been developed for massive and parallel thermodynamic analyses of DNA duplexes. Fluorescently labeled oligonucleotides were hybridized with oligonucleotides immobilized in the 100 x 100 x 20 mum gel pads of the microchips. The equilibrium melting curves for all microchip duplexes were measured in real time in parallel for all microchip duplexes. Thermodynamic data for perfect and mismatched duplexes that were obtained using the microchip method directly correlated with data obtained in solution. Fluorescent labels or longer linkers between the gel and the oligonucleotides appeared to have no significant effect on duplex stability. Extending the immobilized oligonucleotides with a four-base mixture from the 3'-end or one or two universal bases (5-nitroindole) from the 3'- and/or 5'-end increased the stabilities of their duplexes. These extensions were applied to increase the stabilities of the duplexes formed with short oligonucleotides in microchips, to significantly lessen the differences in melting curves of the AT- and GC-rich duplexes, and to improve discrimination of perfect duplexes from those containing poorly recognized terminal mismatches. This study explored a way to increase the efficiency of sequencing by hybridization on oligonucleotide microchips.

Thermodynamic Behavior of Short Oligonucleotides in Microarray Hybridizations Can Be Described Using Gibbs Free Energy in a Nearest-Neighbor Model

“…The fact that the experimental data, when plotted in logarithmic scale, follow a slope that equals 1/RT, implies that the factor a in the above formula equals 1. This is in agreement with the result of Fotin et al 6 on gel pad microarrays for which a = 1.1 ± 0.2. This conclusion should however not be taken as a general statement valid for all types of microarrays.…”

“…In an early experiment on gel pad microarrays, Fotin et al 6 The data presented in this paper allow a determination of the relative differences in ∆G's. The fact that the experimental data, when plotted in logarithmic scale, follow a slope that equals 1/RT, implies that the factor a in the above formula equals 1.…”

“…Whereas the former results were based on the outcome of hybridization experiments only, thermodynamics has been taken into account in several other studies [6][7][8][9][10][11][12][13][14][15][16][17][18][19] . Indeed, hybridization of nucleotide molecules in solution is a well understood biophysical process driven by thermodynamics 20 .…”

“…Fotin et al 6 performed one of the first studies to quantify molecular interaction in a gel pad microarray. They found a convincing linear relationship between microarray hybridization free energies (∆G (chip)) and the corresponding free energies in solution (∆G(solution)).…”

Single Nucleotide Polymorphism Genotyping in Single‐Molecule Electronic Circuits

“…By probing the formation dynamics of probe-target duplexes, the optimal stringency for discriminating SNP alleles can be achieved. [18] In general, molecular beacons display three-phase transitions (i.e., single-stranded state, hairpin state, and probe-target duplex state) upon interacting with target DNAs in solution ( Figure 1b). [13] Because temperature directly determines duplex structure stability in the hairpin stem and probe-target complex, the temperature-dependent profiles of fluorescence fluctuation caused by the concentration differences of the three conformations in solution provide a foundation for previous SNP detection.…”