John M. S. Bartlett.pdf - Bio-Nica.info

256 Case-Green, Pritchard, and Southern
Fig. 1. General scheme for assays using oligonucleotide arrays.
Both methods have advantages and disadvantages, as summarized here.
• In situ fabrication allows a truly combinatorial synthesis of oligonucleotide probes to
be performed, whereas presynthesis involves synthesis of every probe and its individual
application to the array.
• The combinatorial fabrication route may be best suited to producing low numbers or single
copies of many different arrays, whereas presynthesis allows enough pure oligonucleotide
to be produced to make many copies of the same array with speed and reliability.
• Presynthesized oligonucleotides can be purified before addition to the array, whereas
in situ fabrication depends on the reliability and high yields of conventional solid phase
synthesis to ensure that most of the array probe products are correct.
In our laboratory, we have developed barrier-based methods for combinatorial in
situ array synthesis using standard oligonucleotide synthesis reagents (5) confined
within reaction cells (Fig. 2).
These techniques allow combinatorial synthesis of multiple oligonucleotides using
four basic steps (6). Different shapes of cells are used to create arrays with different
organizations and uses. For example, a square-shaped cell can be used for the synthesis
of an array containing overlapping oligonucleotide sequences that have all the possible
complements of a target sequence up to a chosen length of oligonucleotide.
Alternatively, using a block with channels cut into it allows serial synthesis of
oligonucleotides of any sequence in a series of stripes (7,8). If the oligonucleotides
on an array are arranged in parallel lines, it may be used in more than one reaction
if the target samples are hybridized in lines perpendicular to the oligonucleotides (8)
or the array cut into strips.
Assays described in this chapter are performed on amino functionalized polypropylene
support (Beckman) (7,9). This surface is suitable as a support for solid-phase
oligonucleotide synthesis. All syntheses in this chapter use 3′-dimethoxytrityl-5′-N,N′-
diisopropylcyanoethylphosphoramidites (reverse phosphoramidites). Deprotection