Array Design

Microarrays can be made from probes that are either cDNAs or oligonucleotides.

cDNA probes are generated by PCR amplification of cloned cDNA libraries using vector-specific primers, or of genomic DNA using gene-specific primers. Generally, cDNA clones are selected to represent as many unique transcripts as possible. The optimal length for cDNA probes is between 300 – 800 bp. Unfortunately, the amplification steps involved in creating a cDNA probe set are time consuming and carry a high risk of clone contamination.

Using oligonucleotides instead of cDNAs provides a solution to this problem, but at the expense of cost efficiency, and potentially detection sensitivity (Kane et al., 2000) and specificity (Wang et al., 2003). The length for optimal hybridization efficiency varies between 50 and 70bp, although some commercial microarray platforms use oligonucleotides of 25 bp. Designing oligonucleotide probes requires known sequence information, and sequence accuracy is crucial for hybridization efficiency. The probes should contain unique sequences to minimize non-specific cross-hybridization. In contrast to cDNA-based probes, oligonucleotides can be designed to distinguish between alternative spliced transcripts, different alleles, and closely related members of a gene family.

Both cDNA and oligonucleotide probes need to be purified to remove enzymes, nucleotides, buffer salts and detergents, all of which interfere with hybridization. Many vendors sell ready-to-print oligo sets which are already purified.

References:

Kane, M. D., Jatkoe, T. A., Stumpf, C. R., Lu, J., Thomas, J. D., and Madore, S. J. (2000). Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays. Nucleic Acids Res 28, 4552-4557.

Wang, H. Y., Malek, R. L., Kwitek, A. E., Greene, A. S., Luu, T. V., Behbahani, B., Frank, B., Quackenbush, J., and Lee, N. H. (2003). Assessing unmodified 70-mer oligonucleotide probe performance on glass-slide microarrays. Genome Biol 4, R5.