Microfluidic Tools for DNA Analysis - DNA Nanotechnology: From Structure to Function - page 129

7.3 Quantitative Analysis

Complementary to the qualitative analysis, quantitative analysis for DNA molecules
has been widely studied by using microfluidic devices. There research rendered
many interesting applications, relying on unique microfluidic phenomena.

7.3.1 Counting

Micro-well-based methods provide a high-throughput platform that cannot only
be utilized in sequencing but also be introduced into many applications requiring
massive data. For an increasing interest in absolute quantification of DNA copy
number, digital amplification on a chip as well as corresponding facile dispensing
methods for small amount of liquid into micro-wells (or in other word, partitioning)
were developed. Instead of using complicated real-time fluorescent quantitative
PCR or using spectrophotometry that intrinsically requires pure DNA samples with
high concentrations, digital amplification has become an attractive technique to
quantify absolute number (whether high or low) of DNA molecules in an unknown
sample. For a sample with known concentration (i.e., the copy number per unit
volume), the sample can be diluted and distributed into many small compartments
such that on average each compartment contains less than one template DNA;
further, the number of compartments that show positive amplification equals the
number of templates. For a sample with unknown concentration, existing reports
based on Poisson and binomial statistics detailed the calculation process for the
absolute quantification from the fraction of number of compartments that show
positive signal [ 72 - 75 ]. Primarily, the mathematical base of Poisson statistics
requires an as large number of the small compartments as possible [ 76 ]. On
the other hand, however, the large number of compartments will result in large
sample consumption, which is costly and undesirable. Microfluidics handling nano-
[ 74 ], pico- [ 73 ], or even femtoliter [ 77 ] of liquids provides a convenient tool
to accomplish this goal, while shrinking the size of the micro-wells creates the
possibility of the use of samples without further dilution, which could potentially
prevent pipetting errors [ 74 ]. Hansen and coworkers performed digital PCR in
a microfluidic chip consisting of 10 6 wells of picoliter volume with density of
4,400 wells per mm 2 [ 73 ]. Huang and coworkers further expanded the density
up to 20,000 per mm 2 , each with femtoliter volume [ 77 ]. The physically isolated
compartments can be created by pneumatic pressure (Fig. 7.13 )[ 78 ], hydraulic
pressure (Fig. 7.14 )[ 77 ], oil sealing (Fig. 7.15 )[ 79 , 80 ], or slipping (Fig. 7.16 )
[ 81 ]. Basically, they utilized surface tension to retain aqueous solutions into the
wells. To simplify the fluid operation, Mu and coworkers drew lessons from an
automatic introduction technique invented by Hosokawa et al. [ 82 ] and proposed a
smart self-priming compartmentalization strategy to introduce reaction liquids with
uniform nanoliter volume into each micro-well without any external pumps [ 80 ].