Biomedical Engineering Reference
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sticky ends of ( ga ) and the hairpin ( A ) generate the complex ( P ), which includes the
binding site for Fok I. Scission of the complex P regenerates ( ga ) and Fok I, and the
component ( A ) is cleaved into fragments that lack duplex stabilization because of a
low contact of complementary bases. The released single-stranded nucleic acid ( Q )
may be employed as an effector unit that triggers a secondary device, for example,
a G-quadruplex self-assembled from a single-stranded DNA that acts as an aptamer
for thrombin.
14.3.2
Nicking Enzyme-Assisted DNA Machine
Nicking enzyme is a special kind of restriction enzyme. A nicking enzyme (or
nicking endonuclease) is an enzyme that cuts one strand of a ds DNA at a specific
striction site. Such enzymes hydrolyze, cut, only one strand of the DNA duplex, to
produce DNA molecules that are “nicked” rather than cleaved.
The ability of nicking enzymes cutting ss-DNA was first used to design an
isothermal strand displacement amplification (SDA) by Malinowsk in 1992 [ 26 ].
Briefly, the method exploits the strand displacement activity of exo-Klenow to
generate target DNA copies with defined 5 0 and 3 0 ends. The new target generation
process occurs at a single temperature (after initial heat denaturation of the double-
stranded DNA). The target copies generated by this process are then amplified
directly by SDA. The new protocol improves overall amplification efficiency.
Amplification efficiency is also enhanced by improved reaction conditions that
reduce nonspecific binding of SDA primers. Greater than 10 7 -fold amplification of
a genomic sequence from Mycobacterium tuberculosis is achieved in 2 h at 37 ı C.
Shank et al. then carefully studied the kinetic and limiting step of the SDA reaction
[ 27 ]. They found that the SDA reaction is an exponential amplification process, and
the reaction rate is limited by the competition amplifications between target DNA
and other interference DNA.
Willner's group referenced this SDA concept and designed a series of PCR-
mimicking DNA amplification machine [ 28 ]. The autonomous synthesis of the
DNAzyme is depicted in Fig. 14.5 . The template 1, consisting of three regions,
is used as the “track” on which the autonomous synthesis of the DNAzyme is
activated. Region I (orange) is complementary to the primer. The segment II (red) is
complementary to a nucleic acid that, upon hybridization, yields a double strand that
binds the N.BbvC IA nicking endonuclease. Segment III (green) is complementary
to the DNAzyme that is synthesized by the machine. Upon the hybridization of the
primer 2, and in the presence of exonuclease-free Klenow (Klenow fragment, exo-)
polymerase and the nucleotide mixture (deoxynucleotide triphosphate (dNTPs) act
as fuel), the machine is activated. The polymerase-induced reaction replicates the
template. Replication of the template, however, yields the double-stranded domain
that associates Nb.BbvC IA and results in the nicking (scission) of the replicated
single strand at the marked position. The cleavage of the single strand generates
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