Comprehensive viral oligonucleotide probe design using conserved protein regions

Jabado, Omar J.; Liu, Yang; Conlan, Sean; Quan, P. Lan; Hegyi, Hédi; Lussier, Yves; Briese, Thomas; Palacios, Gustavo; Lipkin, W. I.

We pursued experiments to determine the effects of probetarget mismatch and background nucleic acid concentration on array sensitivity and specificity; results were used to derive parameters for probe design. West Nile virus RNA (WNV, strain New York 1999, AF202541) was used as template in hybridization experiments on an Agilent oligonucleotide array with 1131 complementary probes of length 60 nucleotides (nt). Approximately one third of the probes had between 1 and 20 randomly introduced mismatches. The plus and minus (reverse complement) strands of each sequence were deposited, in duplicate. In addition to the flaviviral specific probes, the array contained nearly 36 500 probes for other viral families, negative and positive controls. A volume containing 10 6 copies of WNV and 200 ng of background nucleic acid (human lung tissue RNA) was amplified using random primers and hybridized in four replicate experiments as previously described (10) .