Limits...
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.

Show MeSH

Related in: MedlinePlus

Experimental results of a real-time DNA microarray system using Cy3 and BHQ2 as the donor and acceptor moieties according to method A of Figure 3. In (A), selected fluorescent images (every 30min) are shown during the first 3 h of the hybridization step when 20 ng/100 µl of Target A is introduced to the array. In (B), we show the time series acquired fluorescent light intensity and the calculated average percentage of the occupied probes for four identical capturing spots within this array which have Probe A printing concentration of 10 µM. In (C), we show similar data as of (B), but for capturing spots which have Probe A printing concentration of 2 µM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2802827&req=5

Figure 4: Experimental results of a real-time DNA microarray system using Cy3 and BHQ2 as the donor and acceptor moieties according to method A of Figure 3. In (A), selected fluorescent images (every 30min) are shown during the first 3 h of the hybridization step when 20 ng/100 µl of Target A is introduced to the array. In (B), we show the time series acquired fluorescent light intensity and the calculated average percentage of the occupied probes for four identical capturing spots within this array which have Probe A printing concentration of 10 µM. In (C), we show similar data as of (B), but for capturing spots which have Probe A printing concentration of 2 µM.

Mentions: In Figure 4, we illustrate the experimental results of our real-time method for a typical DNA microarray system. The donor and acceptor are Cy3 and BHQ2, respectively, with Förster distance of ∼6 nm. The sequences of the three different sequences of printed oligonucleotides are listed in Table 1 and they all are printed in four replicates from a solution at 10 and 20 µM concentration. At 100% coupling efficiency such concentrations will create probe surface densities of 1 × 1011 to 2 × 1011 oligonucleotides per mm2 (33,51). For example, a 50 µm diameter microarray spot contains approximately 0.2 × 109 to 0.4 × 109 oligonucleotide molecules for printing concentrations of 10–20 µM.Figure 4.


Real-time DNA microarray analysis.

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

Experimental results of a real-time DNA microarray system using Cy3 and BHQ2 as the donor and acceptor moieties according to method A of Figure 3. In (A), selected fluorescent images (every 30min) are shown during the first 3 h of the hybridization step when 20 ng/100 µl of Target A is introduced to the array. In (B), we show the time series acquired fluorescent light intensity and the calculated average percentage of the occupied probes for four identical capturing spots within this array which have Probe A printing concentration of 10 µM. In (C), we show similar data as of (B), but for capturing spots which have Probe A printing concentration of 2 µM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Experimental results of a real-time DNA microarray system using Cy3 and BHQ2 as the donor and acceptor moieties according to method A of Figure 3. In (A), selected fluorescent images (every 30min) are shown during the first 3 h of the hybridization step when 20 ng/100 µl of Target A is introduced to the array. In (B), we show the time series acquired fluorescent light intensity and the calculated average percentage of the occupied probes for four identical capturing spots within this array which have Probe A printing concentration of 10 µM. In (C), we show similar data as of (B), but for capturing spots which have Probe A printing concentration of 2 µM.
Mentions: In Figure 4, we illustrate the experimental results of our real-time method for a typical DNA microarray system. The donor and acceptor are Cy3 and BHQ2, respectively, with Förster distance of ∼6 nm. The sequences of the three different sequences of printed oligonucleotides are listed in Table 1 and they all are printed in four replicates from a solution at 10 and 20 µM concentration. At 100% coupling efficiency such concentrations will create probe surface densities of 1 × 1011 to 2 × 1011 oligonucleotides per mm2 (33,51). For example, a 50 µm diameter microarray spot contains approximately 0.2 × 109 to 0.4 × 109 oligonucleotide molecules for printing concentrations of 10–20 µM.Figure 4.

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