ENHANCED HIGH-DENSITY OLIGONUCLEOTIDE ARRAY-BASED SEQUENCE-ANALYSIS USING MODIFIED NUCLEOSIDE TRIPHOSPHATES

Pairs of high density oligonucleotide arrays (DNA chips) consisting of >96 000 oligonucleotides were designed to screen the entire 5.53 kb c oding region of the hereditary breast and ovarian cancer BRCA1 gene fo r all possible sequence changes in the homozygous and heterozygous sta tes. Single-stranded RNA targets were generated by PCR amplification o f individual BRCA1 exons using primers containing T3 and T7 RNA polyme rase promoter tails followed by in vitro transcription and partial fra gmentation reactions. Fluorescent hybridization signals from targets c ontaining the four natural bases to >5592 different fully complementar y 25mer oligonucleotide probes an the chip varied over two orders of m agnitude. To examine the thermodynamic contribution of rU.dA and rA.dT target.probe base pairs to this variability, modified uridine [5-meth yluridine and 5-(1-propynyl)-uridine)] and modified adenosine (2,6-dia minopurine riboside) 5'-triphosphates were incorporated into BRCA1 tar gets. Hybridization specificity was assessed based upon hybridization signals from >33 200 probes containing centrally localized single base pair mismatches relative to target sequence. Targets containing 5-met hyluridine displayed promising localized enhancements in hybridization signal, especially in pyrimidine-rich target tracts, while maintainin g single nucleotide mismatch hybridization specificities comparable wi th those of unmodified targets.