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Real-time DNA microarray analysis.

Hassibi A, Vikalo H, Riechmann JL, Hassibi B - Nucleic Acids Res. (2009)

Bottom Line: We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics.We demonstrate in both theory and practice that the time-constant of target capturing in microarrays, similar to all affinity-based biosensors, is inversely proportional to the concentration of the target analyte, which we subsequently use as the fundamental parameter to estimate the concentration of the analytes.Furthermore, to empirically validate the capabilities of this method in practical applications, we present a FRET-based assay which enables the real-time detection in gene expression DNA microarrays.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, TX 78712, USA. arjang@mail.utexas.edu

ABSTRACT
We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics. This method, i.e. real-time DNA microarrays, enhances the detection dynamic range of conventional systems by being impervious to probe saturation in the capturing spots, washing artifacts, microarray spot-to-spot variations, and other signal amplitude-affecting non-idealities. We demonstrate in both theory and practice that the time-constant of target capturing in microarrays, similar to all affinity-based biosensors, is inversely proportional to the concentration of the target analyte, which we subsequently use as the fundamental parameter to estimate the concentration of the analytes. Furthermore, to empirically validate the capabilities of this method in practical applications, we present a FRET-based assay which enables the real-time detection in gene expression DNA microarrays.

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Related in: MedlinePlus

Typical binding kinetics in DNA-based biosensors where the total amount of captured analytes increases until there is no free DNA capturing probes, i.e. saturation region. Expected behavior (thick solid line); Experiment (1, thin solid line); Experiment (2, dashed line); Experiment (3, dotted line).
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Figure 2: Typical binding kinetics in DNA-based biosensors where the total amount of captured analytes increases until there is no free DNA capturing probes, i.e. saturation region. Expected behavior (thick solid line); Experiment (1, thin solid line); Experiment (2, dashed line); Experiment (3, dotted line).

Mentions: The binding process (for both specific and non-specific analytes) is a dynamic process that occurs over time and is a function of the analyte diffusion coefficient and concentration, the temperature, solution ionic strength, the density of the capturing probes, and the reaction surface-to-volume ratio. In Figure 2, we have illustrated a typical dynamical process for an individual capturing process where the total number of captured analytes is denoted by nc(t). It essentially starts with nc(0) = 0, and monotonically increases until it reaches the biochemical steady state, i.e. where the capturing and release processes have equal rate, thus making constant.Figure 2.


Real-time DNA microarray analysis.

Hassibi A, Vikalo H, Riechmann JL, Hassibi B - Nucleic Acids Res. (2009)

Typical binding kinetics in DNA-based biosensors where the total amount of captured analytes increases until there is no free DNA capturing probes, i.e. saturation region. Expected behavior (thick solid line); Experiment (1, thin solid line); Experiment (2, dashed line); Experiment (3, dotted line).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2802827&req=5

Figure 2: Typical binding kinetics in DNA-based biosensors where the total amount of captured analytes increases until there is no free DNA capturing probes, i.e. saturation region. Expected behavior (thick solid line); Experiment (1, thin solid line); Experiment (2, dashed line); Experiment (3, dotted line).
Mentions: The binding process (for both specific and non-specific analytes) is a dynamic process that occurs over time and is a function of the analyte diffusion coefficient and concentration, the temperature, solution ionic strength, the density of the capturing probes, and the reaction surface-to-volume ratio. In Figure 2, we have illustrated a typical dynamical process for an individual capturing process where the total number of captured analytes is denoted by nc(t). It essentially starts with nc(0) = 0, and monotonically increases until it reaches the biochemical steady state, i.e. where the capturing and release processes have equal rate, thus making constant.Figure 2.

Bottom Line: We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics.We demonstrate in both theory and practice that the time-constant of target capturing in microarrays, similar to all affinity-based biosensors, is inversely proportional to the concentration of the target analyte, which we subsequently use as the fundamental parameter to estimate the concentration of the analytes.Furthermore, to empirically validate the capabilities of this method in practical applications, we present a FRET-based assay which enables the real-time detection in gene expression DNA microarrays.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cellular and Molecular Biology, University of Texas at Austin, TX 78712, USA. arjang@mail.utexas.edu

ABSTRACT
We present a quantification method for affinity-based DNA microarrays which is based on the real-time measurements of hybridization kinetics. This method, i.e. real-time DNA microarrays, enhances the detection dynamic range of conventional systems by being impervious to probe saturation in the capturing spots, washing artifacts, microarray spot-to-spot variations, and other signal amplitude-affecting non-idealities. We demonstrate in both theory and practice that the time-constant of target capturing in microarrays, similar to all affinity-based biosensors, is inversely proportional to the concentration of the target analyte, which we subsequently use as the fundamental parameter to estimate the concentration of the analytes. Furthermore, to empirically validate the capabilities of this method in practical applications, we present a FRET-based assay which enables the real-time detection in gene expression DNA microarrays.

Show MeSH
Related in: MedlinePlus