can be fit on a microarray. Noncontact printing has been used in one
method of in situ synthesis (Figure 10.24c). In this method, all bases are
deprotected followed by site-specific dispensing of phosphoramiditeprotected nucleosides; cycles of deprotection and the addition of nucleosides are repeated to complete oligonucleotide synthesis. Perhaps the most
practical methods for fabrication of DNA microarrays are the printing
methods in which all oligonucleotides are synthesized beforehand. Via
contact or noncontact printing, known quantities of each individual oligonucleotide are dispensed at their desired position (Figure 10.24d).
10.7.3 Optimization of DNA microarrays
A great deal of optimization is required before a DNA microarray can be
used effectively. The quality of a DNA microarray is greatly influenced by
the method in which it was fabricated. For example, spot density, the
number of spots that can be placed in a given area of a microarray, varies
greatly among in situ and noncontact printing and contact printing
methods. Using in situ methods, spot sizes with diameters less than 10 μm
have been created, whereas printing methods can create spot sizes
between 20 and 30 μm. Also affecting spot density is the array geometry. It is
important for spots to be arranged in a geometry that maximizes spot
density while preventing any overlap; additionally, precise array geometry
is significant for future data analysis. Currently, our ability to create high
spot density arrays has surpassed our ability to accurately interpret fluorescence. Current fluorimeters are capable of resolving fluorescence from
spots as small as 30 μm. The development of higher resolution fluorimeters
will be required for further miniaturization of DNA microarrays.
Spot morphology is another parameter that must be monitored. A
homogeneous spot is preferred for data analysis. Poor spot morphology is
less of an issue with light-directed in situ syntheses. Spot morphology of
printing methods can be improved by controlling humidity and temperature as well as by adjusting the spotting buffer.
Perhaps the most important factors to be controlled for optimization are
probe density and hybridized density. These two factors are closely
related, as probe density is the number of probes in a given spot and
hybridized density is the fraction of these probes that hybridize when
saturated with target molecules. In order to get the greatest fluorescent
signal, hybridized density must be maximized; however, a number of
issues arise. If probe density is too high, probe molecules may interact
with other probe molecules on the surface and become unable to
DNA AND LIPID MICROARRAYS 423
method of in situ synthesis (Figure 10.24c). In this method, all bases are
deprotected followed by site-specific dispensing of phosphoramiditeprotected nucleosides; cycles of deprotection and the addition of nucleosides are repeated to complete oligonucleotide synthesis. Perhaps the most
practical methods for fabrication of DNA microarrays are the printing
methods in which all oligonucleotides are synthesized beforehand. Via
contact or noncontact printing, known quantities of each individual oligonucleotide are dispensed at their desired position (Figure 10.24d).
10.7.3 Optimization of DNA microarrays
A great deal of optimization is required before a DNA microarray can be
used effectively. The quality of a DNA microarray is greatly influenced by
the method in which it was fabricated. For example, spot density, the
number of spots that can be placed in a given area of a microarray, varies
greatly among in situ and noncontact printing and contact printing
methods. Using in situ methods, spot sizes with diameters less than 10 μm
have been created, whereas printing methods can create spot sizes
between 20 and 30 μm. Also affecting spot density is the array geometry. It is
important for spots to be arranged in a geometry that maximizes spot
density while preventing any overlap; additionally, precise array geometry
is significant for future data analysis. Currently, our ability to create high
spot density arrays has surpassed our ability to accurately interpret fluorescence. Current fluorimeters are capable of resolving fluorescence from
spots as small as 30 μm. The development of higher resolution fluorimeters
will be required for further miniaturization of DNA microarrays.
Spot morphology is another parameter that must be monitored. A
homogeneous spot is preferred for data analysis. Poor spot morphology is
less of an issue with light-directed in situ syntheses. Spot morphology of
printing methods can be improved by controlling humidity and temperature as well as by adjusting the spotting buffer.
Perhaps the most important factors to be controlled for optimization are
probe density and hybridized density. These two factors are closely
related, as probe density is the number of probes in a given spot and
hybridized density is the fraction of these probes that hybridize when
saturated with target molecules. In order to get the greatest fluorescent
signal, hybridized density must be maximized; however, a number of
issues arise. If probe density is too high, probe molecules may interact
with other probe molecules on the surface and become unable to
DNA AND LIPID MICROARRAYS 423