Biomedical Engineering Reference
In-Depth Information
hybridization processes on microbead surfaces and (ii) for multiplex melting curve
analysis in a future real-time VideoScan assay format.
6.4 Multiplex Hybridization Temperature Optimization
on a Microbead Surface
Diagnostic applications based on nucleic acid hybridization require rapid reaction
kinetics to deliver fast results, especially in a clinical context. To achieve fine
tuning of the critical parameters, a multiplex approach is favorable. One of the
main strength of VideoScan is its ability to assess kinetics of multiplexed samples
both in solution and on the surface of microbeads.
The rate, stringency and efficiency of a hybridization reaction are crucial for
optimal assay development and performance. To investigate multiplex hybrid-
ization events in solution, the number of available FRET pairs or filter sets limits
the multiplexing level (e.g., in qPCR cyclers). The DNA hybridization kinetics for
spatially resolved microbeads was done under precisely controlled temperature
conditions and high microbead redundancy (compare [
80
,
81
] for planar arrays).
VideoScan can be used to study the multiplex hybridization at customizable
temperatures, either in solution or solid phase under identical conditions requiring
only one FRET pair. During each measurement cycle, microbeads are tracked
individually. Our aim was to determine the optimal hybridization temperature in a
multiplex format.
The Atto 647N fluorescence dye-labeled capture probes (Fig.
7
) SERCA2-cap,
GAPDH-cap, and MLC-2v-cap (Table
8
) were coupled to microbeads according to
Armstrong et al. [
24
]. Prepared microbeads were pooled in 20 lL PCR buffer [
3
]
(2.5 mM MgCl
2
) including quencher-labeled target probes for GAPDH-cap and
MLC-2v-cap at a final concentration of 55 nM and sealed with 25 lL mineral oil.
The VideoScan HCU was set to denature the samples for 30 s at 95 C and to
subsequently heat the samples at a defined temperature. Measurement was done at
indicated time intervals.
Two capture probes (MLC-2v-cap, GAPDH-cap) were tested for optimal
hybridization conditions in the presence of control for non-specific hybridization
(SERCA2). It was found that the optimal hybridization temperature is close to
50 C for MLC-2v-cap and GAPDH-cap (Fig.
21
). Both capture probes had similar
hybridization efficiencies of 75% but MLC-2v-cap reached its plateau after 20 min
while GAPDH-cap needed more than 60 min hybridization time. Despite the fact
that a higher hybridization temperature of 65 C initially resulted in a quicker
hybridization, the final hybridization efficiency was drastically lower (*20%) than
at 50 C. This behavior is expected due to the start of melting processes (compare
Fig.
20
for MLC-2v-cap). The negative control SERCA2-cap remained unchanged.
