Biology Reference
In-Depth Information
DAQ-Electronics
Controller PC
Servo controller
Piezo electric
nozzles
Microchip
FIGURE 1.2
DNA microchip synthesis with piezoelectric inkjet technology. The microarray synthesis platform consists of a controller
PC,DAQ-electronics,andaservocontroller, which together control the movement and the printing of the inkjet print head
onto slide surfaces. Droplets are dispensed onto as many as 10 000 spot features that are each less than 100 μmin
diameter.
channel. In this work, the discrete features on the substrate could hold and segregate 100 pL
droplets using pre-patterned surface tension (
Fig. 1.2
).
9
Piezoelectric print head technology is primarily commercialized by Agilent. In 2001,
Hughes et al. used Agilent
s second-generation system for gene-expression profiling of
biological samples from various organisms.
39
The flexibility of the inkjet system allowed
them to create new arrays and update sequences rapidly. Since then, gene expression
profiling has been conventionally performed on Agilent microarrays.
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42
Through
refinement of surfaces, firing parameters, and solvent mixtures, Agilent has evolved its
22 575 feature throughput to a current 244 000 feature throughput on a single glass slide.
Their current systems now also include computer visualization systems, print head
maintenance modules, and automated slide handling.
'
GENE ASSEMBLY
Chemical oligonucleotide synthesis accumulates errors due to side reactions and
inefficiencies in the stepwise reactions.
43
Although oligonucleotides of up to 600 bp
in length can be synthesized, yields are extremely low.
44
Gene assembly becomes
necessary for synthesis of longer constructs. Almost all current assembly techniques use
a combination of PCR or ligation-based assembly. The advantage of these methods
over restriction digestion/ligation methods is that they can perform scarless and
sequence-independent assembly.
Ligation-based assembly uses thermo-stable DNA ligase to join pre-synthesized
oligonucleotides. Successful use of ligation-based assembly has been demonstrated in
research and commercial synthesis platforms.
45
47
Thermo-stable ligase is advantageous
compared to T4 DNA ligase, because fewer DNA secondary structures will form at elevated
ligation temperatures.
48
50
Ligation-based assembly involves ligation and PCR amplification.
Overlapping oligonucleotides are designed to completely cover both strands of the sequence.
They are phosphorylated at the 5
0
-ends for the ligation reaction. The oligonucleotides
