An Affymetrix GeneChip is a DNA oligonucleotide microarray, which contains hundreds of thousands to millions of distinct 25-mer oligonucleotides. Each oligonucleotide sequence is present in millions of copies in a specific area, termed “probe cell”. To profiling gene expression, representative messenger RNA sequences are first selected based on GenBank, EST and RefSeq databases. A probe set, which contains 11 to 20 probe pairs (25-mer oligonucleotides for each probe sequence), is then designed to interrogate expression of a messenger RNA sequence. To avoid the problem of cross hybridization, each probe pair contains a perfect match sequence and a mismatch sequence, which are randomly spaced throughout the entire array. The expression level of a targeted messenger RNA is calculated according to signal differences between perfect match sequences and mismatch sequences for the coordinated probe set (gene).
The Affymetrix array technology employs an in situ synthesis of hundreds of thousands to millions of distinct 25-mer oligonucleotides onto glass surface using photolithography and combinatorial chemistry. Up to 1.3 million different DNA oligonucleotide probes are simultaneously synthesized on an array in a series of cycles. In brief, a glass substrate is first coated with linkers containing photolabile protecting groups. A mask is then applied to protect selected probe cells and expose the rest of the cells to ultraviolet light. Illumination removes the photolabile protecting groups and hence enables selective nucleoside phosphoramidite addition only at the previously exposed sites. Next, a different mask is applied and the cycle of illumination and chemical coupling is performed again. By repeating this cycle, a specific set of oligonucleotide probes is synthesized with each probe type in a known location. The completed probe arrays are packaged into cartridges.
In a microarray experiment, a probe is defined as a unique DNA sequence in situ synthesized or printed on an array, such as 25-mer oligonucleotides on the Affymetrix platform, or double-stranded cDNA and long oligonucleotides spotted on a glass slide or a nylon filter membrane. A target is defined as a labeled cDNA or cRNA “targets” made from total RNA of your samples.
