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 coding region of the hereditary breast and ovarian cancer BRCA1 gene for all possible sequence changes in the homozygous and heterozygous states. Single-stranded RNA targets were generated by PCR amplification of individual BRCA1 exons using primers containing T3 and T7RNA polymerase promoter tails followed by in vitro transcription and partial fragmentation reactions. Fluorescent hybridization signals from targets containing the four natural bases to >5592 different fully complementary 25mer oligonucleotide probes on the chip varied over two orders of magnitude. To examine the thermodynamic contribution of rU.dA and rA.dT target.probe base pairs to this variability, modified uridine [5-methyluridine and 5-(1-propynyl)-uridine)] and modified adenosine (2,6-diaminopurine riboside) 5'-triphosphates were incorporated into BRCA1 targets. 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-methyluridine displayed promising localized enhancements in hybridization signal, especially in pyrimidine-rich target tracts, while maintaining single nucleotide mismatch hybridization specificities comparable with those of unmodified targets.
Hacia
J G
JG
National Human Genome Research Institute, National Institutes of Health, Building 31, Room 4B09, 31 Center Drive, Bethesda, MD 20892-2152, USA.
Woski
S A
SA
Fidanza
J
J
Edgemon
K
K
Hunt
N
N
McGall
G
G
Fodor
S P
SP
Collins
F S
FS
D020137:Base Pair Mismatch; D001483:Base Sequence; D001943:Breast Neoplasms / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics; D005260:Female; D019398:Genes, BRCA1; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D066298:In Vitro Techniques; D009693:Nucleic Acid Hybridization; D009711:Nucleotides; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D010051:Ovarian Neoplasms / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D014158:Transcription, Genetic
D017931:DNA Primers; D004273:DNA, Neoplasm; D009711:Nucleotides; D015345:Oligonucleotide Probes; D012334:RNA, Neoplasm