Enhanced high density oligonucleotide array-based sequence analysis using modified nucleoside triphosphates.
AUTOR(ES)
Hacia, J G
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=147927Documentos Relacionados
- Application of High-Density Array-Based Signature-Tagged Mutagenesis To Discover Novel Yersinia Virulence-Associated Genes
- High-Throughput Analysis of Subtelomeric Chromosome Rearrangements by Use of Array-Based Comparative Genomic Hybridization
- Detection of Salmonella enterica Serovar Typhimurium by Using a Rapid, Array-Based Immunosensor
- 1-Mb Resolution Array-Based Comparative Genomic Hybridization Using a BAC Clone Set Optimized for Cancer Gene Analysis
- SBE-TAGS: An array-based method for efficient single-nucleotide polymorphism genotyping