Feng, Xiaodong_ Xie, Hong-Guang - Applying pharmacogenomics in therapeutics-CRC Press (2016)

40 Applying Pharmacogenomics in Therapeutics
After amplification, the PCR-positive (1 template) and PCR-negative (0 template)
partitions are counted to provide an absolute quantification of the starting DNA
copies in digital form (Figure 2.1d).
How to efficiently capture or isolate individual molecules is the major determinant
for performing a high-quality digital PCR. The first commercial system for
digital PCR was introduced by Fluidigm (www.fluidigm.com) in 2006 based on integrated
fluidic circuits (chips) composed of chambers and valves to partition samples
(Heyries et al. 2011). Droplet Digital PCR technology is another method of partitioning
innovated by QuantaLife (now part of Bio-Rad [www.biorad.com]). This
method separates a DNA template into 20,000 nanoliter-sized droplets for individual
PCR reactions and provides digital counting of each target (Hindson et al. 2013).
RainDance Technologies further reduced the size of the droplets, leading to the generation
of up to 10 million picoliter-sized droplets per assay (http://raindancetech.
com/digital-pcr-tech/).
Given that digital PCR technology could detect DNA molecules with additional
sensitivity, accuracy, and precision, it has many applications. Digital PCR can be
used in the detection and quantification of rare genetic sequences, CNVs, single-cell
genetic variations, and rare pathogens. Moreover, sample preparation in many NGS
platforms, including Roche/454, ABI/SOLiD, and Life Technologies/Ion Torrent,
is enabled by single-step digital PCR as a key factor to reduce the time and cost
(Sandberg et al. 2011; Williams et al. 2006).
Microarray Technology
Although PCR is a widely used and easily applied method for analyzing genetic
variations, the number of primers that can be mixed together and annealed to different
target sequences is limited. To fulfill the need for throughput increase, the
technology of microarray, a multiplex lab-on-a-chip, was demonstrated by Schena
et al. (1995). Since then, several companies, including Affymetrix, Agilent, Applied
Microarrays, NimbleGen, and Illumina, have greatly facilitated the expansion of the
microarray technology. Microarray technology fixes designed single-stranded DNA
probes on a solid substrate, for example, a glass slide or silicon thin-file cell, and processes
with the biological specimen for hybridization or capture of the probes’ targets
(complementary DNA or RNA) (Figure 2.2). It relies on the base-pairing principle
that nucleic acids bind to their complementary strands (A to T and G to C) to differentiate
targets with different sequences. The target samples are usually labeled with
fluorescence or other chemiluminescent molecules, and hybridization to the probes
can be detected by specialized equipment. This allows high-throughput screening of
target samples with miniaturized, multiplexed, and parallel processing and detection.
The throughput has increased dramatically in past decades from roughly 400 bacterial
gene targets manually handled in a 1982 study (Augenlicht and Kobrin 1982) to
approximately 2 million genome-wide human SNPs coupled with automatic scanning
and image processing recently (Genome-Wide Human SNP Array 6.0, Affymetrix
[www.affymetrix.com/catalog]). These changes allow DNA microarrays to be used
for broad applications that include the measurement of gene expression levels, detection
of SNPs, and targeted resequencing on a genome-wide scale.