Biomedical Engineering Reference
In-Depth Information
Ref . electrode
-
-
-
A
A
A
A
C G
T
GC
AT
C G
TA
GC
V G
-
C
T
G
C
C
T A
GC
A
T
-
GC
Si 3 N 4
Si 3 N 4
Si 3 N 4
SiO 2
Si
Si 3 N 4
SiO 2
Si
Si 3 N 4
SiO 2
Si
SiO 2
Si
SiO 2
Si
e -
e -
e -
e -
e -
e -
e -
e -
e - e -
e -
e -
e -
e -
e -
e -
e -
e -
e -
e -
e -
e -
e -
e -
e -
initial
dCTP
dATP
dGTP
dTTP
iterative addition
Fig. 6.17
Principle of label-free DNA sequencing based on intrinsic molecular charges
Tabl e 6. 2
Base length of probe and target DNA
Locus
Function
Sequence
R353Q
R353Q-wild type
probe
5 0 -amino group-CCACTACCG -3 0 (9mer)
5 0 -ACGTGCCCCGGTAGTGG -3 0 (17mer)
target
122
122
wild type
5 0 -amino group-CGTCCTCTGAA-3 0 (11mer)
probe
5 0 -AGCTGGGGTGTTCAGAGGACG-3 0 (21mer)
target
C282Y
C282Y-wild type
probe
5 0 - amino group-AGATATACGTG-3 0 (11mer)
5 0 -CTCCACCTGGCACGTATATCT-3 0 (21mer)
target
buffer concentration to be used for measuring charge density change at the gate
surface. The potential change induced by adsorption of proteins at the gate
surface was reported to be dependent on the electrolyte concentration [ 25 ]. It is
therefore important to optimize the Debye length at the gate insulator/solution
interface. In the present study, a 0.025 M phosphate buffer solution was used for
measuring charge density change at the gate surface, while the conventional reaction
mixture was used for single-base extension reaction.
The 11-base oligonucleotide probes were immobilized on the gate surface and
hybridized with the 21-base target DNA for the base sequence of -122 (Table 6.2 )
[ 18 ]. We evaluated the FETs in combination with single-base extension for DNA
sequencing. We prepared four kinds of buffer solution containing both DNA poly-
merase and one of dCTP, dATP, dGTP, or dTTP, respectively. The FETs hybridized
with target DNA were immersed into the conventional reaction solutions for single-
base extension reaction and the V T shift was measured in a 0.025 M phosphate
buffer solution after washing the FETs. The cycle of single-base extension and
measurement of the V T was repeated iteratively to determine the base sequence of
the target DNA. When the base sequence of R353Q region of the factor VII gene was
used as a target DNA, the V T shifted in the positive direction only after single-base
 
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