Biology Reference
In-Depth Information
Microarrays are 2D solid-phase arrays used to assay or screen biological materials like
nucleic acids, proteins, or cells. Oligonucleotide arrays can be used for a variety of designs,
including gene expression screens, SNP genotyping, comparative genomic hybridization
(CGH), tiling, ChIP-on-chip, microRNA, resequencing, and aptamer screening.
8
More
relevantly, oligonucleotide microarrays can offer significant reductions in DNA synthesis
cost due to their dense and massively parallel feature designs. For example, reducing costs
by scaling down reagent volumes in resin-based synthesis is restricted to decreasing the
diameter of capillaries. Simply eliminating the rinsing of lines in instrumentation makes
at least a 10-fold reduction in cost.
6
This is because acetonitrile is often used in high
volumes for rinsing and dissolving reagents, making it one of the most expensive bulk
reagents.
9
Cost for reagents in a custom inkjet microarray slide is less than USD50 per slide,
due to the low volumes of phosphoramidite and tetrazole necessary for the miniaturized
platform.
10
Microarray synthesis not only lowers costs, but also makes synthesis more
environmentally friendly. High-density arrays can offer 10
4
10
6
unique oligonucleotides,
and can reduce costs by at least an order of magnitude.
11,12
The price for 3912 90-mers
from LC Sciences is about USD1000; the Agilent 55 k chip,
USD7000, which translates to
USD0.0025 per base.
13
These costs do not include downstream costs for gene assembly.
B
The variety in technologies and techniques for microarray-based oligonucleotide synthesis
is expected to offer reductions in cost and improvements in throughput in the coming years.
In contrast, the cost of column-synthesized oligonucleotides has remained constant over
the past six years and is unlikely to decrease significantly.
14
The microarray technologies
that exist in the DNA synthesis market include ink-jet printing (Agilent, Protogene),
photosensitive 5
0
deprotection (Nimblegen, Affymetrix, Flexgen), photo-generated acid
deprotection (Atactic/Xeotron/Invitrogen, LC Sciences), and electrolytic acid/base arrays
(Oxamer, Combimatrix/Customarray).
5
Optimizations that have been made to microarray synthesis include synthesis on
PDMS (poly(dimethylsiloxilane))
15
and COC (cyclic olefin copolymer)
16
substrates as
low-cost and flexible alternatives to glass. Ma and coworkers showed that oxCOC, a hybrid
substrate composed of COC and RF sputtered SiO
2
, takes benefits from both constituents.
17
COC offers low density, resistance to organic solvents, high stiffness, and UV transparency,
while SiO
2
offers useful surface linkers for phosphoramidite chemistry. Furthermore,
oxCOC can be manipulated for large-scale production. With soft lithography, a PDMS
stamp can be made to imprint channels or wells on COC before thin-film deposition
of SiO
2
.
18
The stamps are disposable and can be created in a non-cleanroom setting with
silicon molds, although the silicon molds must be fabricated via photolithography. A study
involving inkjet microchip synthesis demonstrated that using a COC chip with patterned
silica features reduced the error rate of synthesized oligonucleotides from one in 200 bases
to one in 600 bases,
16
which is equivalent to high-quality column synthesis.
The dominant costs are now enzymatic processing, cloning, and sequencing.
12
A recent
study by Tian
s group addresses the limitations of processing steps using an integrated
combination of isothermal nicking, strand displacement amplification (nSDA), and
polymerase cycling assembly (PCA), reducing the cost even further to USD0.005 per base
with an error frequency of
'
0.2 errors/kb.
19
Such reduced cost could make gene and
gene library synthesis more widely accessible.
,
Microfluidic and Fluidic Systems
Microfluidics allows for the control and manipulation of small volumes of liquids.
These features make it particularly useful for a variety of applications like PCR and cell
screening. More specifically, microfluidics can decrease the space and cost requirements
of DNA synthesis. A recent study reports a programmable microfluidic synthesis platform
that can synthesize
B
100 pmol of each unique oligonucleotide, which is substantial
