Probe array features

Figure 3. Probe array features examined in this study, (a) Fluorescence microscope image and map of probe types and base substitution sequences, (b) Illustration of the tiling for the 10 sequence positions within a given set of probes. We have used the lO-mer probes for illustration matched positions are in bold-face type.

The number of probe sites (features) per unit surface area in a DNA array reflects its information density and versatility in terms of parallel analysis of different sequences. In order to maximize these parameters, the features and their spacing in the array should be as small as possible, while retaining full sensitivity and discrimination in terms of detection. Decreasing the size of the features has the additional advantage of reducing the amount of target sample required for analysis in the application. 

A new method for the removal of the 5 -0-DMT group during the synthesis of ODNs on a microarray has been studied" which uses all the steps involved in phosphoramidite synthesis, but uses a photogenerated acid (PGA) rather than trichloroacetic acid to deprotect. The photolabile 3 -0- [2-(2-nitrophenyl)propoxy]carbonyl -protected 5 -phosphoramidites (5) have been prepared for the 5 ->3 light-directed synthesis of DNA on microarrays." Developments for the synthesis of high-density DNA probe arrays employ nucleoside monomers protected with 5 -(a-methyl-6-nitropiperonyloxycarbonyl) (MeN-POC) with proximity photolithography, which is currently capable of printing 10 m probe features at a density of 10 probes/cm. "... 

X 1.28 cm probe array, with individual 18 x 18 micron features, and 16 probe pairs per probe set, can interrogate over 20,000 transcripts. This number is steadily increasing as manufacturing improvements shrink the feature size, and... 

Nitrocellulose and nylon membranes have been widely used in the production of macroarrays (arrays with probe sites of diameter 0.5 to 1 mm), but not so much in the production of microarrays (feature size of 25 to 200 xm) because of a lack of spot resolution (see Sect. 5.2, Spotting of Capture Probes). These membranes exhibit lateral wicking characteristics and the probe therefore tends to spread out from the point of apphcation. Casting of these membranes onto the surface of glass slides is a solution to this problem [28]. 

Other terms also relate to the anatomy of the microarray (Figure 1.4). The probes are immobilized on the substrate at discrete (x, y) locations or spatially addressable sites. The probe spots [measured in microns (diameters) for a circular spotted array or as a side of a square for an in situ array] are often referred to as features or elements of an array. Thus, an array containing 10,000 features would have 10,000 probes arranged as an array on a substrate. 

Nimblegen adopted the MAS approach to create arrays containing as many as 786,432 probe features in a 2.3-cm area on a slide. Oligonucleotides in the range of 24 to 90-mer are synthesized with good stepwise yields. In order to accommodate current scanner resolution, the densities have been reduced. Two formats covering the area of a standard slide are provided 195,000 probes (1 4) and 390,0(X) probes (1 2). A 24-mer array can be synthesized within 2 to 4 hr. The average stepwise yield is 97.5%. 

Wodicka et al. (1997) created such an array for measuring yeasf gene expression based upon 25-mer oligonucleotides covering 6200 ORFs. Each ORF was represented by 20 PM and 20 MM probes. Why so many probes Simply put, not all probes hybridize in a predictable manner. Averaging across a number of probes improves the outcome. Thus, the yeast expression chip comprised over 65,000 probe features and required a set of four chip subarrays. 

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