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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

Capturing curves and the computed time-constants from a real-time DNA microarray system for 2 ng/100 μl and 20 ng/100 μl analyte concentrations.
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Figure 5: Capturing curves and the computed time-constants from a real-time DNA microarray system for 2 ng/100 μl and 20 ng/100 μl analyte concentrations.

Mentions: In Figure 5, we show the real-time results of four microarray experiments, with the same target and probe sequence (i.e. Probe A and Target A), but different concentrations. Each curve is generated using the results of eight independent spots on the array. Based on Equation (9), τ should be proportional to and inversely proportional to nt. As evident, this indeed the case, i.e. doubling the probe density doubles the time-constant and reducing the target concentration by one order of magnitude decreases the time-constant by one order of magnitude. We should note that the estimation of τ is robust with respect to the probe printing variations and artifacts. Namely, the coefficients of variation of the initial probe light intensity in probe spots with printing concentrations of 10 and 20 µM were 22 and 15%, respectively. Nevertheless, coefficients of variation of the corresponding estimates of τ were 6 and 4.4% in the experiment with 20 ng/100 µl of the target, and 4.8 and 2.1% in the experiment with 2 ng/100 µl of the target, respectively.Figure 5.


Real-time DNA microarray analysis.

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

Capturing curves and the computed time-constants from a real-time DNA microarray system for 2 ng/100 μl and 20 ng/100 μl analyte concentrations.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 5: Capturing curves and the computed time-constants from a real-time DNA microarray system for 2 ng/100 μl and 20 ng/100 μl analyte concentrations.
Mentions: In Figure 5, we show the real-time results of four microarray experiments, with the same target and probe sequence (i.e. Probe A and Target A), but different concentrations. Each curve is generated using the results of eight independent spots on the array. Based on Equation (9), τ should be proportional to and inversely proportional to nt. As evident, this indeed the case, i.e. doubling the probe density doubles the time-constant and reducing the target concentration by one order of magnitude decreases the time-constant by one order of magnitude. We should note that the estimation of τ is robust with respect to the probe printing variations and artifacts. Namely, the coefficients of variation of the initial probe light intensity in probe spots with printing concentrations of 10 and 20 µM were 22 and 15%, respectively. Nevertheless, coefficients of variation of the corresponding estimates of τ were 6 and 4.4% in the experiment with 20 ng/100 µl of the target, and 4.8 and 2.1% in the experiment with 2 ng/100 µl of the target, respectively.Figure 5.

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