Biomedical Engineering Reference
In-Depth Information
10
4
F=m
2
for the genetic FET, the amount of
charges increased after hybridization is calculated to be 5:1
Since V
T
D
12 mV and Ci
D
4:3
10
6
C=m
2
. The base
lengths of the oligonucleotide probe and the target DNA used in the experiment
are both 17 bases, which correspond to 5.78 nm in length. Negative charges derived
from phosphate groups are distributed along the double-stranded DNA from the
gate surface to the bulk of the sample solution. We assume that these negative
charges along the DNA molecules contributed to the V
T
shift equally and that
all the oligonucleotide probes were hybridized with the target DNA. Under these
assumptions, the number of oligonucleotide probes on the channel region can be
calculated to be 2:3
10
8
=cm
2
. The surface density
of oligonucleotide probes immobilized on glass, silicon dioxide, and gold has been
reported to be in the order of 10
9
10
4
, which corresponds to 1:9
10
13
=cm
2
, which was determined by different
methods [
19
-
24
]. Since the density of the oligonucleotide probes is strongly
dependent on the method and materials used for a substrate and immobilization,
the number of oligonucleotide probes immobilized on silicon nitride could be
increased by optimizing the immobilization method. It is noted that hybridization
with 2:3
10
4
target DNA molecules resulted in the V
T
shift of 12 mV. Therefore,
detection of DNA molecules by the use of the genetic FET can be very sensitive, if
hybridization is carried out sufficiently.
6.4.1.3
Label-Free DNA Sequencing Based on Intrinsic
Molecular Charges
Although a number of methods for single nucleotide polymorphism (SNP) anal-
ysis have been developed as described in the previous works, DNA sequencing
techniques are still required to be improved in terms of cost, simplicity, and high
throughput in order to analyze not only SNPs but also genomic variations such as
insertion/deletion and short tandem repeat. A new method for DNA sequencing is
introduced in this section, which is based on detection of intrinsic charges of DNA
molecules using the field effect.
Oligonucleotide probes are immobilized on the Si
3
N
4
gate surface. The com-
plementary target DNA is hybridized with the oligonucleotide probes on the
gate surface. The hybridization events are followed by the introduction of DNA
polymerase and one of each deoxynucleotide (dCTP, dATP, dGTP, or dTTP).
DNA polymerase extends the immobilized oligonucleotide probes in a template-
dependent manner (Fig.
6.17
). As a result of extension reaction, negative charges
increase at the gate surface of the FETs because of intrinsic negative charges of
incorporated molecules. This charge density change can be detected as a shift of the
V
T
of the FETs. Thus, iterative addition of each deoxynucleotide and measurement
of V
T
allow a direct, simple, and nonlabeled DNA sequencing.
The base sequences of factor VII gene including two SNP sites and that of
hereditary hemochromatosis (Table
6.2
)[
18
] were used to demonstrate the principle
of DNA sequencing based on the FETs. We have paid special attention to the
