Enhanced high density oligonucleotide array-based sequence analysis using modified nucleoside triphosphates

Abstract

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.

A System for Specific, High-throughput Genotyping by Allele-specific Primer Extension on Microarrays

“…In contrast, our method requires only two probes per mutation and thus can be set up with a simple spotting robot. The poor specificity of ASO-based microarray systems in certain sequence contexts is a well-recognized problem, and a variety of strategies have been employed to overcome this problem, such as an electronic stringency changer (Gilles et al 1999), the use of modified bases in the targets and the inclusion of chaotropic agents in the buffer (Hacia et al 1998b;Nguyen et al 1999), and the monitoring of melting curves of the hybrids (Drobyshev et al 1997). The high fidelity of the RT enzyme in our system ensures good genotype discrimination at a single set of reaction conditions.…”

Preparation of Oligonucleotide Arrays with High-Density DNA Deposition and High Hybridization Efficiency

“…The labeled oligonucleotide was purified through a G50 column before use. The oligonucleotide-spotted slides were dried at room temperature for about 16 h. The slides were washed with water, 3 M NH 4 OH, and finally a Wash Buffer I (1× SSPE, 0.2% SDS) to clearly remove unbound oligonucleotides. For quantification of immobilization, the labeled oligonucleotide was used and the amount of the immobilized oligonucleotide was quantitated by analyzing the slide with a Molecular Dynamics PhosphoroImager.…”

“…With the development of array technology, a number of solid supports and various attaching methods have been published. [1][2][3][4][5][6][7] Glass slides are generally used as standard supports for making microarray. The deposition of pre-fabricated nucleic acids and direct in situ synthesis on the solid surface are prevailing protocols to produce DNA array.…”

Unrestricted accessibility of short oligonucleotides to RNA

“…Higher-order structures of RNA (and to a lesser extent single-stranded DNA) have severely impeded sequence detection by short probes of 8-10 nucleotides (nt) in length (Doty et al 1959;Lima et al 1992;Hacia et al 1998). This limitation has prevented their general use as specific agents for detection of single nucleotide polymorphisms and point mutations (Wallace et al 1979;Zoller and Smith 1983), and has posed a serious block to the development of universal arrays for application in the resequencing of genes or the profiling of genetic samples.…”