Examination of fluid carry-over in micro channel
Beads were saturated with non-complementary BP-2, and mixed with various
concentrations of target ssDNA. Without any incubation, bead isolation was performed and
solution in W2 was collected. Supernatant in W2 was subsequently measured for fluorescent
signals and converted for concentrations. Percentage of molecule carry-over was calculated by
dividing concentration measured in W2 over initial concentration in W1. Note that target ssDNA
was served merely as an indicator of fluid, thus incubation was not designed.
Effect of location of BP targeting sequence in target ssDNA
We investigated potential effect of location at which biotinylated capture probe bind to
model ssDNAs on the bead separation efficiency. Three similar sequences of ssDNA (t1-t3) with
the biotinylated capture probe’s binding site shifted along 3’ end to 5’ end by 12-15 nucleotides
each (bold and underlined) were designed (Table I).
Table I. Sequences of model ssDNA.
Oligo
Sequence
Length
(nt)
74
5’- /56-FAM/ aaaa CAGGGTTAAAG AAGAACAAA TCGGT
ATGGGTGCGAGAGCGTCAATATTAAG AACAG aaaaaaaaaa
aaaa-3’
5’- /56-FAM/ aaa a CAGGGTTAAAG ATGGGTGCGA
ssDNA test2
74
GAGCGTCAATATTAAG AAGAACAAATCGGT AACAG
(t2)
aaaaaaaaaa aaaa-3’
5’- /56-FAM/ aaa ATGGGTGCGAGAGCGTCAATATTAAG
ssDNA test3
74
(t3)
ACAGGGTTAAAG AAGAACAAATCGGTAACAG aaaaaaaaaa
aaaa-3’
Thermodynamic calculations for hybridization events between BP and target ssDNAs were
ssDNA test1
(t1)
performed, as shown in figure 1 and table II, using the DINAmelt and mFold Web Server
software. As expected, delta G is more negative at lower hybridization temperature, where
higher temperature brings up the energy to break the hybridization bonds. For specific
hybridization events 1 to 4, since the hybridization regions are identical, we calculated similar Tm
values at around 67˚C, which is much higher than that of the non-specific hybridization event 5.
FIG. 1. Thermodynamically simulated hybridization structures of ssDNA sequences and BP at room
temperature (25°C), [Na+]=150mM, [Mg2+]=20mM with 5nM concentration. BP is highlighted in cyan
shadow. A) BP hybridizes with target ssDNA. B) BP hybridizes with model ssDNA t1. C) BP hybridizes
with model ssDNA t2. D) BP hybridizes with model ssDNA t3.
Table II. Theoretical hybridization energies between BP and ssDNA.
Hybridiz
ation
Event
1
2
3
4
5
The
Oligonucleotide(s)
Delta G
Value
(25°C)
Delta G
Value
(41°C)
Delta G
Value
(60°C)
Delta G
Value
(70°C)
Tm
(°C)
Target ssDNA + biotinylated
-39.0
-30.0
-19.3
-13.7
67.8
capture probe
ssDNA t1 + biotinylated
-39.5
-30.1
-19.0
-13.3
66.9
capture probe
ssDNA t2 + biotinylated
-37.7
-28.8
-18.5
-13.0
66.5
capture probe
ssDNA t3 + biotinylated
-38.3
-29.3
-18.7
-13.6
66.9
capture probe
Target ssDNA + biotinylated
-11.1
-5.9
-3.0
-2.2
<20
capture probe-2
model ssDNAs and biotinylated capture probe were mixed at 1:1 ratio in hybridization
buffer and incubated for 20 min at room temperature. The hybridization products were identified
by running gel electrophoresis (Agilent Small RNA kit). Figure 2a shows the result of a small
RNA gel listing the sizes of each component in nucleotide length. All the ssDNAs showed sizes
around 65nt (before hybridization), where the shorter linear length could result from the RNA
secondary structure in buffer. Biotinylated capture probe showed 26nt in length as expected. In
the mixture of ssDNA and biotinylated capture probe, only one band was seen at the ssDNA
length (~65nt), suggesting complete hybridization between biotinylated capture probe and
ssDNAs. Figure 2b shows that the three different hybridization products have the same
concentration. Hence the formation of three hybridized ssDNA and biotinylated capture probe
complexes are determined to be identical.
FIG. 2. (a) A small RNA gel plot showing specificity of binding between target ssDNA and biotinylated
capture probe. ssDNA test sequence t1, t2 and t3 were examined. Lane1: ssDNA test1; Lane2: ssDNA
t1 + biotinylated capture probe at 1:1 ratio; Lane3: biotinlyated capture probe; Lane4: ssDNA test2; Lane5:
Target ssDNA t2 + biotinylated capture probe at 1:1 ratio; Lane6: biotinlated capture probe; Lane7:ssDNA
test3; Lane8: ssDNA t3 + biotinylated capture probe at 1:1 ratio; Lane9: biotinlated capture probe. Missing
gel bands in lanes 2, 5, and 8 for 26nt suggest confirmed hybridization of biotinylated capture probe with
target ssDNA. All pseudo gel electrophoresis was generated by a 2100 Bioanaylzer and a small RNA gel
cartridge (Agilent, CA).
(b) Electropherogram concentration profiles of lane 2, 5 and 8.
Effect of incubation temperature on hybridization of target ssDNA
Target ssDNA needs to diffuse from the bulk volume to the surface in order to interact
with complementary biotinylated capture probe on the bead surface1. This is also evidenced by
the fact that a mixing procedure that involves rotating the device reduced the hybridization time
from overnight to 3 hours2. To optimize the hybridization efficiency, we compared hybridized
target ssDNA concentration on bead surface under different incubation temperatures (room
temperature, 40 ºC and 70 ºC) with fixed incubation time (total 15min). We find that bead
captured most target ssDNA when incubated at 40 ºC (figure 3).
FIG. 3. Effect of incubation temperature on target ssDNA hybridization on bead surface. Concentration of
target ssDNA bond to bead is shown as number of ssDNA per unit area (μm 2) on bead surface.
1
2
R. Drmanac. DNA-Sequence Determination by Hybridization - a Strategy for Efficient LargeScale Sequencing (Vol 260, Pg 1649, 1993). Science 263, 596-596, (1994).
M. Chee, R. Yang, E. Hubbell, A. Berno, X. C. Huang, D. Stern, J. Winkler, D. J. Lockhart, M. S.
Morris & S. P. Fodor. Accessing genetic information with high-density DNA arrays. Science
274, 610-614, (1996).