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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

Two FRET-based real-time DNA microarray assaying alternative methods. In method (A) the donor fluorophore is attached to the capturing probe, while in method (B) it is placed near the capturing probe by attaching it to a ‘dummy’ probe. In both methods, successful hybridization of the analyte which contains the quencher results in the quenching of the nearby donor fluorophore.
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Figure 3: Two FRET-based real-time DNA microarray assaying alternative methods. In method (A) the donor fluorophore is attached to the capturing probe, while in method (B) it is placed near the capturing probe by attaching it to a ‘dummy’ probe. In both methods, successful hybridization of the analyte which contains the quencher results in the quenching of the nearby donor fluorophore.

Mentions: To enable real-time detection in microarrays with little background interference, we need to ensure that only the captured analytes in intimate proximity of the capturing probes contribute to the measured signal. Since intimate proximity in molecular scale in individual spots is critical, here we propose to use fluoresce resonance energy transfer (FRET) moieties to create binding-specific signals (25–30). In this technique, we attach radiating donor molecules (e.g. fluorescent molecules) to the capturing probes (method A in Figure 3) or to a ‘dummy’ probe near the capturing probes (method B in Figure 3) in each spot. This can be done prior to array spotting and during the synthesis of the capturing probes. For instance, in the case of DNA microarrays, as shown in Figure 3, the DNA oligonucleotides that act as the capturing probes are end-labeled with Cyanine (Cy) fluorophores. Subsequently, in the sample preparation process, we attach the acceptor molecules of the FRET system to the analytes. If the sample containing the analytes is applied to the array, which consists of capturing spots with donors, hybridization events bring the donor and acceptor into intimate proximity resulting in a molecular FRET system. To create a binding-specific signal, we use non-radiating acceptors (i.e. quenchers), such that hybridization ‘turns off’ the fluorophore of the capturing probe or the ‘dummy’ probe, and hence reduces the overall emitted fluorescent signal of the spot as shown in Figure 3. From a imaging point of view, this method requires identical instrumentation compared to other fluorescence-based assays, while the solution containing the sample introduces little fluorescence background. In addition, parallel measurements can be carried and the method is scalable to large size arrays. It is also important to recognize that the low background signal in this method enables the effective detection of the capturing events with high SNR during the DNA hybridization.Figure 3.


Real-time DNA microarray analysis.

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

Two FRET-based real-time DNA microarray assaying alternative methods. In method (A) the donor fluorophore is attached to the capturing probe, while in method (B) it is placed near the capturing probe by attaching it to a ‘dummy’ probe. In both methods, successful hybridization of the analyte which contains the quencher results in the quenching of the nearby donor fluorophore.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Two FRET-based real-time DNA microarray assaying alternative methods. In method (A) the donor fluorophore is attached to the capturing probe, while in method (B) it is placed near the capturing probe by attaching it to a ‘dummy’ probe. In both methods, successful hybridization of the analyte which contains the quencher results in the quenching of the nearby donor fluorophore.
Mentions: To enable real-time detection in microarrays with little background interference, we need to ensure that only the captured analytes in intimate proximity of the capturing probes contribute to the measured signal. Since intimate proximity in molecular scale in individual spots is critical, here we propose to use fluoresce resonance energy transfer (FRET) moieties to create binding-specific signals (25–30). In this technique, we attach radiating donor molecules (e.g. fluorescent molecules) to the capturing probes (method A in Figure 3) or to a ‘dummy’ probe near the capturing probes (method B in Figure 3) in each spot. This can be done prior to array spotting and during the synthesis of the capturing probes. For instance, in the case of DNA microarrays, as shown in Figure 3, the DNA oligonucleotides that act as the capturing probes are end-labeled with Cyanine (Cy) fluorophores. Subsequently, in the sample preparation process, we attach the acceptor molecules of the FRET system to the analytes. If the sample containing the analytes is applied to the array, which consists of capturing spots with donors, hybridization events bring the donor and acceptor into intimate proximity resulting in a molecular FRET system. To create a binding-specific signal, we use non-radiating acceptors (i.e. quenchers), such that hybridization ‘turns off’ the fluorophore of the capturing probe or the ‘dummy’ probe, and hence reduces the overall emitted fluorescent signal of the spot as shown in Figure 3. From a imaging point of view, this method requires identical instrumentation compared to other fluorescence-based assays, while the solution containing the sample introduces little fluorescence background. In addition, parallel measurements can be carried and the method is scalable to large size arrays. It is also important to recognize that the low background signal in this method enables the effective detection of the capturing events with high SNR during the DNA hybridization.Figure 3.

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