incubated with the surface and chemical coupling occurs only at those sites that have been irradiated in the
preceding step. Light is next directed at further regions of the substrate by a new mask, and the reaction
sequence is successively repeated for the second, third and fourth of the four monomers, A, C, G and T to
complete the first cycle. A second complete cycle lays down the second nucleotide in the oligomer. Further
repetitions of this cycle provide the full set of 4Npolydeoxyribonucleotides of length N, or any subset, in
just Ncomplete cycles. Thus, for a given reference sequence, a DNA array can be designed that consists
of a highly dense collection of DNA single-stranded oligomers, usually around 25 residues long. This
photolithographic process enables construction of arrays with extremely high information content. Large-
scale commercial methods permit approximately 300,000 oligodeoxynucleotides to be synthesised on
small 1.281.28 cm arrays, while versions with 10^6 probes per array are being developed.
In a separate development, Patrick Brown at Stanford University developed a cDNA spotting method that
is suited to the display of single-DNA fragments, often greater than several hundred base pairs in length.^21
cDNA samples (about 15 ng) are micro-spotted robotically onto a glass (or nylon membrane) surface that has
been treated chemically to provide primary amino groups. Droplets (1nL) are located 200
m apart and
the DNA in the spots is covalently bonded to the surface by UV irradiation to link the surface amino
groups to thymidine residues.
A third robotic methodology has been developed by Rosetta Inpharmatics that uses ink-jet printer tech-
nology to perform classical oligodeoxyribonucleotide synthesis based on the four-step dimethoxytrityl pro-
tecting group chemistry (Section 4.1.2).^22 In a fourth approach, Agilent Labs in collaboration with Marvin
Caruthers have developed a two-step microarray synthesis cycle to halve the number of steps required to
186 Chapter 5
Figure 5.14 (a) Deoxythymidine 3-phosphoramidite with a 5-photolabile -methyl-2-nitropiperonyloxycarbonyl
protecting group. (b) dUTP analogue linked through the 5-methyl group to a cyanine dye having red
fluorescence emission. (c) Deoxyuridine 3-phosphoramidite linked via C-5 to a protected fluorescein
dye having green light emission