DNA Microarrays

The development of ''DNA microarrays'' or generally named ''DNA chips'' has been driven by modern approaches to analyze multiple gene mutations and expressed sequences [2]. The broad range of actual DNA chip applications includes the detection of pathogens, the measurement of differences in the expression of genes between different cell populations, and the analysis of genomic alterations

Fig. 6.2.1. Fabrication of an array by spotting or spatially addressable synthesis, probing with the analyte, and readout (modified after Ref. [1]).

(e.g. single nucleotide polymorphisms). In general the promise of DNA micro-arrays for cell biologists is to provide a more complete molecular view of cellular states and responses in complex tissues.

The analyte is most often fluorescence-labeled DNA or RNA molecules isolated from a cell extract. After hybridization with oligonucleotides immobilized on the microarray a fluorescence image can be generated which can be compared with one of an extract of cells in a different state (Figure 6.2.2). The underlying assumption is that genes at an upregulated level result in an increase in mRNA, which can be monitored by using this technology.

The generation of DNA microarrays can be achieved in two different ways: on the one hand one can immobilize either cDNA, generated by biochemical techniques from mRNA, or synthetic oligonucleotides on a glass slide using chemical functionalities introduced during conventional automated synthesis [3, 4]. These linking units - added at the 3' end (starting point of the synthesis), internal positions, or the 5' end (end of the synthesis) - can react with complementary functionalities on the glass surface (Table 6.2.1). Synthesis of the desired oligonucleotides directly on a glass slide is an alternative way in preparing arrays. One must distinguish between two different protecting group strategies:

2) labelling

1) DNA isolation

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