7,859 research outputs found
Real-time DNA microarray analysis
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
Modeling the kinetics of hybridization in microarrays
Conventional fluorescent-based microarrays acquire data
after the hybridization phase. In this phase the targets analytes
(i.e., DNA fragments) bind to the capturing probes
on the array and supposedly reach a steady state. Accordingly,
microarray experiments essentially provide only a
single, steady-state data point of the hybridization process.
On the other hand, a novel technique (i.e., realtime
microarrays) capable of recording the kinetics of hybridization
in fluorescent-based microarrays has recently
been proposed in [5]. The richness of the information obtained
therein promises higher signal-to-noise ratio, smaller
estimation error, and broader assay detection dynamic range
compared to the conventional microarrays. In the current
paper, we develop a probabilistic model of the kinetics of
hybridization and describe a procedure for the estimation
of its parameters which include the binding rate and target
concentration. This probabilistic model is an important
step towards developing optimal detection algorithms for
the microarrays which measure the kinetics of hybridization,
and to understanding their fundamental limitations
A novel cassette method for probe evaluation in the designed biochips
A critical step in biochip design is the selection of probes with identical hybridisation characteristics. In this article we describe a novel method for evaluating DNA hybridisation probes, allowing the fine-tuning of biochips, that uses cassettes with multiple probes. Each cassette contains probes in equimolar proportions so that their hybridisation performance can be assessed in a single reaction. The model used to demonstrate this method was a series of probes developed to detect TORCH pathogens. DNA probes were designed for Toxoplasma gondii, Chlamidia trachomatis, Rubella, Cytomegalovirus, and Herpes virus and these were used to construct the DNA cassettes. Five cassettes were constructed to detect TORCH pathogens using a variety of genes coding for membrane proteins, viral matrix protein, an early expressed viral protein, viral DNA polymerase and the repetitive gene B1 of Toxoplasma gondii. All of these probes, except that for the B1 gene, exhibited similar profiles under the same hybridisation conditions. The failure of the B1 gene probe to hybridise was not due to a position effect, and this indicated that the probe was unsuitable for inclusion in the biochip. The redesigned probe for the B1 gene exhibited identical hybridisation properties to the other probes, suitable for inclusion in a biochip
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