Affymetrix - GeneChip® Probe Selection and Array Design


		Probe Design and Selection

		Probe Design

		Every probe on an Affymetrix microarray is designed to determine whether or not the complementary sequence of RNA or DNA is present in the sample. At the molecular level, the probe must distinguish one sequence from billions of similar sequences. Affymetrix probes are designed for this explicit purpose. Usually 25 nucleotides in length (25-mer), Affymetrix probes have high specificity and are better designed to reject targets that are not identical. This level of specificity is essential when measuring the expression of two very similar gene sequences and is critical when genotyping DNA, as researchers need to distinguish between four nearly-identical sequences that differ in only one base. The high density of GeneChip microarrays affords the ability to use multiple probes for each expression or genotype measurement made. Just as the 25-mer probe length confers high specificity, the use of multiple probes provides for high sensitivity and reproducibility; 22 probes are routinely used for each expression measurement and 40 for each genotype call. The probe set–a combination of 25mer probes–strikes the most optimal balance between sensitivity and specificity, allowing for consistent discrimination between signal and background noise and accurate data sets. For each probe on the array that perfectly matches its target sequence, Affymetrix also builds a paired “mismatch” probe. The mismatch probe contains a single mismatch located directly in the middle of the 25-base probe sequence. While the perfect match probe provides measurable fluorescence when sample binds to it, the paired mismatch probe is used to detect and eliminate any false or contaminating fluorescence within that measurement. The mismatch probe serves as an internal control for its perfect match partner because it hybridizes to nonspecific sequences about as effectively as its counterpart, allowing spurious signals, from cross hybridization for example, to be efficiently quantified and subtracted from a gene expression measurement or genotype call. Read more about the perfect match / mismatch strategy in this technical note (pdf, 82 KB).

		Probe Selection

		Probe selection and array design strategies are core elements that result in the reliability, sensitivity, specificity, and versatility of GeneChip® microarrays. Stringent probe selection strategies ensure that the best probes for each transcript or sequence to be analyzed are included in the array. For each, transcript or DNA sequence to be interrogated, multiple probes that uniquely represent that sequence or transcript are selected. Millions of raw sequences and SNPs from multiple databases in the public domain are screened. This results in probes with the desired intensity and relative concentration-dependence, with minimal cross-hybridization. Strategies to optimize probe hybridization are invariably included in the process of probe selection. All probe sequence descriptions and annotations are openly accessible through the NetAffx Analysis Center, which allows a seamless connection between array design and downstream analysis.