3A26D227d01 - review 20 nature genetics supplement •...

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Unformatted text preview: review 20 nature genetics supplement • volume 21 • january 1999 Biological systems read, store and modify genetic information using the rules of molecular recognition. Every nucleic acid strand carries the capacity to recognize complementary sequences through base pairing. The process of recognition, or hybridization, can be highly parallel; every sequence in a complex mixture can, in principle, be interrogated simultaneously. We have used these simple principles to develop powerful new experimental tools designed to collect and analyse vast amounts of genetic and cellu- lar information. The introduction, development and integration of two key technologies 1–5 form the cornerstone of the new meth- ods. The first is the fabrication of hundreds of thousands of polynucleotides at high spatial resolution in precise locations on a surface. The second, laser confocal fluorescence scanning, facili- tates the measurement of molecular binding events on the array. These technologies and some variants have been adopted in both the commercial and academic sectors (see pages 25 (ref. 6), 10 (ref. 7) and 15 (ref. 8) of this issue). At Affymetrix, we have focused on light-directed synthesis for the construction of high-density DNA probe arrays using two techniques: photolithography and solid-phase DNA synthesis. We attach synthetic linkers modified with photochemically remov- able protecting groups to a glass substrate and direct light through a photolithographic mask to specific areas on the surface to pro- duce localized photodeprotection (Fig. 1). The first of a series of chemical building blocks, hydroxyl-protected deoxynucleosides, is incubated with the surface, and chemical coupling occurs at those sites that have been illuminated in the preceding step. Next, light is directed to different regions of the substrate by a new mask, and the chemical cycle is repeated 9,10 . Highly efficient strategies can be used to synthesize arbitrary polynucleotides at specified locations on the array in a minimum number of chemi- cal steps 1 . For example, the complete set of 4 N polydeoxynu- cleotides of length N, or any subset, can be synthesized in only 4 × N cycles. Thus, given a reference sequence, a DNA probe array can be designed that consists of a highly dense collection of com- plementary probes with virtually no constraints on design para- meters. The amount of nucleic acid information encoded on the array in the form of different probes is limited only by the physical size of the array and the achievable lithographic resolution. Current large scale commercial manufacturing methods allow for approximately 300,000 polydeoxynucleotides to be synthesized on small 1.28 × 1.28 cm arrays—experimental versions now exceed one million probes per array....
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3A26D227d01 - review 20 nature genetics supplement •...

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