Oligonucleotide probes synthesis

Probe arrays and DNA target. The studies described here have been carried out with arrays of 10-, 12-, 14-, 16-, 18-, and 20-base oligonucleotide probe synthesis sites 200 jim X 200 jim in size. The probes are covalently attached to a siloxane derivatized 15 mm X 15 mm x 0.7 mm borosilicate glass substrate and were synthesized using photolithographic techniques that have been described elsewhere. U... 

Cyanine dyes also are used as labels for oligonucleotide probes. Unlike the hydrophilic cyanine dyes valuable for protein labeling, the use of dye-phosphoramidite compounds to synthesize DNA or RNA probes typically requires the use of more hydrophobic dye structures to make them compatible with the solvents and reactions of oligonucleotide synthesis. Thus, indol cyanines containing few or no sulfonates are used in these applications to label oligos for applications such as array detection, hybridization assays, and RT-PCR. 

FIGURE 11.2 Photolithographic synthesis of oligonucleotide probe arrays. 

Direct protein sequencing of the large proteins which constituted the channel was not possible both because of the very limited amount of material available and because of the not unexpected very hydrophobic nature of these membrane proteins. However short sequences from the amino terminal were obtained and these were sufficient to allow synthesis of degenerate oligonucleotide probes which were then used to probe a library of cDNA synthesized from a small quantity of mRNA extracted from the electric organ of the eel. The cDNA from the positive plasmids identified in this way was then sequenced and the primary amino acid sequence of all the subunits so obtained. 

As the density of information derived from efforts to sequence, map and identify human genes increased, so did the demand for analytical tools capable of exploiting this information. DNA microarrays were developed in response to this demand. Southern(69) was the first to describe parallel, in situ ohgonucleotide synthesis as a means of generating oligonucleotide probe arrays on solid supports for highly parallel hybridization analysis. Southern s method uses standard nucleotide synthetic reactions to synthesize the oligonucleotides. The reactions are carried out in a movable chamber, which provides a physical barrier between the reaction chamber and the intended synthesis area. 

FIGURE 23.1 Schematic representation of genome-scale gene expression analysis with DNA microarrays, (a) DNA microarrays produced by probe deposition (b) Oligonucleotide microarrays produced by in situ probe synthesis (Affymetrix technology). 

A last possibility to obtain probes grafted onto beads is to directly synthesize the oligonucleotide probes onto glass beads via phosphoramidite reaction [18] (Fig. 6). The synthesis reaction sequence is composed of a coupling step between a protected nucleotide immobilized onto the bead and a tetra-zole activated and protected nucleotide (step C). This is followed by a capping and a de-blocking of the newly added nucleotide (step D and B). The cycle is then repeated until the desired sequence is obtained. 

A plethora of methods are now available for labelling oligonucleotide probes for hybridisation experiments. Many of these procedures are compatible with automated DNA synthesis and the introduction of multiple reporter groups provides a means of amplifying the signal from the probe. 

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