Biomedical Engineering Reference
In-Depth Information
Fig. 16 Homogeneous microbead hybridization probe assay. a A single-stranded hybridization
capture probe labeled at its 3
0
end with Atto 647N is bound via biotin to streptavidin-coated
microbeads. The ''detector probe'' is labeled with a black hole quencher ''Q'' (BHQ2) at its 5
0
end. In the absence of PCR product hybridization, the capture probe and detector probe are kept
in solution and FRET does not occur. b A FRET signal is only detectable when hybridization of
all three molecules occurs due to an increase of the amount of PCR product
useful tool for semi-automated mPCR studies. We use this assay routinely in a
96-well format for medium-throughput gene analysis with high sensitivity and
specificity. The analysis of more than 400 E. coli strains is an ongoing project.
5.2 Homogeneous Microliter-Volume Hybridization Using
Advalytix
Slides
Rapid and sensitive detection of DNA targets is crucial in many disciplines
of nucleic acid analysis (e.g., gene expression, single nucleotide polymorphism).
For analytic platforms, the detection limit is found to be one of the most critical
parameters. We applied a homogeneous multiplex oligonucleotide hybridization
assay based on FRET to determine the lowest detectable oligonucleotide con-
centration. Since VideoScan can process multiple well geometries, we utilized
the AmpliGrid system (AG480F, Advalytix
, Beckman Coulter) and performed a
homogeneous
multiplex
direct
hybridization
assay
(Fig.
7
)
and
microbead
hybridization probe assay (Fig.
16
) in a volume as small as 1 lL.
Three microbead populations were loaded with capture probes GAPDH, VIM
and aCS-cap (Table
8
) as described in
Sect. 3.2
. GAPDH was included as negative
control for non-specific quenching. The microbead populations were pooled and
adjusted to 20-50 microbeads per lL per population. A droplet of 0.5 lL mi-
crobead suspension was transferred to a spot of an AmpliGrid slide. Further 0.5 lL
aliquots of the sample were added with increasing concentrations, from 0 to 200
nM final concentration, of target molecules for both VIM and aCS-cap. All spots
were sealed with 5 lL mineral oil. The hybridization (2.5 min at 95 C, 30 min
at 50 C, hold at 20 C) was performed in the AmpliSpeed ASN 400 thermal
cycler.
