Biomedical Engineering Reference
In-Depth Information
a
b
Factor VII R353Q
Hereditary hemochromatosis C282Y
TGCACGGG
3'-GCCATCACC-5' (9 bases)
5'-
ACGTGCCC
CGGTAGTGG-3' (17 bases)
GAGGTGGACC
3'-GTGCATATAGA-5' (11 bases)
5'-
CTCCACCTGG
CACGTATATCT-3' (21 bases)
15
13
11
9
7
5
15
13
11
9
7
5
C
AGTCAGTCAGT
C
AGTCAGTCAGT
GGG
GG
GG
CC
T
A
T
G
C
A
C
G
A
3
1
-1
3
1
-1
Fig. 6.18
Label-free DNA sequencing based on intrinsic molecular charges
extension with the specific deoxynucleotides which were complementary to the base
sequence of the target DNA (Fig.
6.18
a). The V
T
change for three-base extension,
GGG, was 6.9 mV, which was bigger than that for one-base extension, but was not
three times as big as that expected from the number of intrinsic charges. Although
the linear relationship between the base length synthesized by the extension reaction
and the V
T
shift was obtained in the range from 0 to 30 bases (Fig.
6.16
), it is
important to detect single-base extension quantitatively, in order to reduce base
call error especially for continuous sequence of the same base. The density and
orientation of the immobilized oligonucleotide probes have to be controlled during
a series of extension reactions at 72
ı
C. Further improvement of precision of the base
call is also expected by automation of extension reaction and VT measurements.
C282Y region of hereditary hemochromatosis gene was used as another example
of DNA sequencing using the FET and single-base extension (Fig.
6.18
b). The
positive V
T
shifts could be detected in accordance with the base sequence of the
target DNA. In this case, the average V
T
shifts for two-base incorporation was
5.8 mV with the standard deviation of 0.4 mV, while the average V
T
shift for single-
base extension was 3.2 mV. The V
T
shifts for two-base extension was approximately
twice as big as that for single-base extension. Thus, the results of iterative extension
reaction and detection of the V
T
indicated the ability of a direct, simple, and
potentially precise DNA sequencing analysis using the FETs. The number of bases
which can be analyzed by the proposed method is about ten bases at present.
The V
T
shift for single-base extension became gradually smaller as the number of
bases increased more than ten bases. One of the reasons for this limitation would
be the Debye length at the gate insulator/solution interface. Any charge density
change induced outside the Debye length cannot be detected with the FETs. Lateral
extension reaction in which DNA probes are extended in parallel with the gate
surface would be effective for DNA sequencing with long bases. Another reason for
the limitation would be peeling off the immobilized oligonucleotide probes from
the surface of the gate insulator as the temperature stress of the extension reaction at
72
ı
C is applied repeatedly. The stronger immobilization method for oligonucleotide
probes on the Si
3
N
4
surface has to be adopted to analyze longer base sequence.
