Biomedical Engineering Reference
In-Depth Information
Fig. 20 Multiplex melting curve and melting peak analysis on six microbead populations.
At about 48 C poly(dT)
20
and poly(dA)
20
start to melt followed by the gene-specific probes at
temperatures above 60 C
surface of microbeads, we employed direct hybridization (Fig.
7
) and hybridiza-
tion probe (Fig.
16
) chemistry. Only one ligand channel was used for interrogation
of the poly(dT)
20
region and the gene-specific regions. Six microbead populations
were loaded with capture probes VIM-cap, MLC-2v-cap, SERCA2-cap, poly(dA)
20
-
cap, aCS-cap, and HPRT1-cap. The poly(dT)
20
regions were interrogated. The
capture probe VIM-cap (Table
8
) was immobilized using the biotin-streptavidin
interaction and the remaining via amide bonds as described in [
3
]. Microbeads
were immobilized as described in
Sect. 3.1
. Targets were hybridized in PCR buffer
(2.5 mM MgCl
2
) as described in
Sect. 5.2
. Excess of non-hybridized target was
removed by washing two times with 100 lL PCR buffer. Samples were added to
20 lL PCR buffer, sealed with 25 lL mineral oil, and placed in the HCU. The
HCU was operated by stepwise heating (1 C/step) between 25 and 95 C with a
dwell time of 15 s. Fluorescence data were processed with RKWard [
27
]as
described in
Sect. 6.2
.
Using the VideoScan HCU it was possible to measure eight melting tempera-
tures on six microbead populations simultaneously. A first melting effect was
measured at about 48 C by a decrease of the refMFI (Fig.
20
)ofpoly(dT)
20
-cap
regions of MLC-2v-cap, SERCA2-cap, aCS-cap, HPRT1-cap. The slight variances
observed are presumably due to the covalent immobilization. The same applied to
the poly(dA)
20
probe where the release of the poly(dT)
20
quencher lead to an
increase of refMFI. As a control, VIM-cap without a poly(dT)
20
-cap region
remained unchanged. With increasing temperature melting of VIM-cap (70.5 C),
MLC-2v-cap (72.5 C), and aCS-cap (74 C) were measured. SERCA2-cap and
HPRT1-cap were left with no complementary quencher oligonucleotide (negative
control) and remained unchanged.
In conclusion, VideoScan HCU is useful for determining melting temperatures
of immobilized DNA fragments and is therefore useful (i) for basic analysis of
