Microfluidic Tools for DNA Analysis - DNA Nanotechnology: From Structure to Function - page 118

beads containing DNA colonies were pick out, and the binding affinity of DNA in
each fluorescent bead against the target molecule is screened via high-throughput
cytometry.

7.2.1.3 Solid-Phase Amplification

The homogeneous reaction is normally performed in tubes or wells, so multi-
plexing can be accomplished by using multiple tubes or wells. By contrast, the
heterogeneous scheme enables the multiple individual reactions to be carried out
within one same area by spatially segregating reactants via immobilization. For the
microfluidic system, the latter could avoid complicated fluid control and therefore
greatly simplify the chip design and external supplies.
The basic principle of the SPA was detailed in 2000 by Adessi et al. [ 28 ]. As
shown in Fig. 7.5 a, two primers used in PCR are 5 0 -end covalently attached to the
glass surface. The template DNA hybridize to the surface-bound primers as normal
PCR, and the primers are elongated from 5 0 to 3 0 with the DNA polymerase to
produce a copy of the hybridized template. This copy is covalently attached to the
surface to which the primer was originally attached, and the surface-bound copies
can also hybridize to attached primers and form additional copies in the vicinity of
the initial copies. The initial templates can be released into the solution again and
again and hybridize other primers to initiate new elongation. The SPA takes place
until the attached primers are saturated.
SPA could intrinsically eliminate the interferences between different primers
(i.e., primer dimers, which could lead to competition with PCR amplification of
targeted DNA sequence, or interference with accurate quantification in real-time
PCR). Based on this approach, a multiplexed reverse transcription PCR (RT-PCR)
in a fluidic chamber was developed [ 30 ]. The researchers attached three pairs of
primers targeting three genes of avian influenza virus (AIV) to the surface and
subtyped AIV (Fig. 7.5 c). Compared to DNA microarray technology based on static
hybridization, multiplexed SPA introduced dynamic polymerization in a microarray
format [ 31 ], resulting in high throughput and high sensitivity. There is a major
difference with the “strict” SPA that has been detailed in Fig. 7.5 a. In the strict
SPA, all primers are attached onto the solid support, and therefore, all amplified
single-strand DNA are also anchored onto the solid support. The strict SPA is less
efficient than conventional solution-based amplification [ 32 , 33 ], which may be
primarily attributed to steric hindrance. To overcome this drawback, a simultaneous
amplification including on-solid support reaction and in-liquid solution reaction was
proposed (Fig. 7.5 b). In this kind of SPA, only one primer is attached onto the
solid support. The freely diffusible primers and resulting amplicons accelerate the
SPA [ 29 ].
The principle of SPA can also be adopted in isothermal amplifications. Westin
and coworkers attached different sets of biotinylated primers targeting different
genes on a streptavidin layer by using the so-called electronic anchoring technique
and performed a multiplexed SDA (strand displacement amplification) for multiple