Genetic studies aimed at understanding the molecular basis of complex human phenotypes require the genotyping of many thousands of single-nucleotide polymorphisms (SNPs) across large numbers of individuals. Public efforts have so far identified over two million common human SNPs; however, the scoring of these SNPs is labor-intensive and requires a substantial amount of automation. Here we describe a simple but effective approach, termed whole-genome sampling analysis (WGSA), for genotyping thousands of SNPs simultaneously in a complex DNA sample without locus-specific primers or automation. Our method amplifies highly reproducible fractions of the genome across multiple DNA samples and calls genotypes at >99% accuracy. We rapidly genotyped 14,548 SNPs in three different human populations and identified a subset of them with significant allele frequency differences between groups. We also determined the ancestral allele for 8,386 SNPs by genotyping chimpanzee and gorilla DNA. WGSA is highly scaleable and enables the creation of ultrahigh density SNP maps for use in genetic studies.

Recent studies of human populations suggest that the genome consists of chromosome segments that are ancestrally conserved ('haplotype blocks'; refs. 1-3) and have discrete boundaries defined by recombination hot spots. Using publicly available genetic markers, we have constructed a first-generation haplotype map of chromosome 19. As expected for this marker density, approximately one-third of the chromosome is encompassed within haplotype blocks. Evolutionary modeling of the data indicates that recombination hot spots are not required to explain most of the observed blocks, providing that marker ascertainment and the observed marker spacing are considered. In contrast, several long blocks are inconsistent with our evolutionary models, and different mechanisms could explain their origins.

The covalent attachment of disulfide-modified oligonucleotides to a mercaptosilane-modified glass surface is described. This method provides an efficient and specific covalent attachment chemistry for immobilization of DNA probes onto a solid support. Glass slides were derivatized with 3-mercaptopropyl silane for attachment of 5-prime disulfide-modified oligonucleotides via disulfide bonds. An attachment density of approximately 3 ؋ 10 5 oligonucleotides/m 2 was observed. Oligonucleotides attached by this method provided a highly efficient substrate for nucleic acid hybridization and primer extension assays. In addition, we have demonstrated patterning of multiple DNA probes on a glass surface utilizing this attachment chemistry, which allows for array densities of at least 20,000 spots/cm 2 . © 1999 Academic Press Key Words: covalent immobilization; oligonucleotide; glass; disulfide bonds; DNA microarray.In recent years, high-density miniaturized oligonucleotide arrays have emerged as promising tools for assessing genomic data with a lower cost and higher throughput than the traditional gel-based methods. Such oligonucleotide arrays, or DNA chips, have been applied to genetic mutational scanning (1, 2), molecular bar coding (3), gene expression monitoring (4, 5), and sequencing (6 -8). The power of the DNA chips come from the highly parallel, addressable, miniaturized array format that provides significant advantages over traditional gel-based formats in terms of reagent cost, labor, speed, throughput, and operational simplicity. The development of efficient chemistries for the manufacture of spatially resolved, microscale DNA arrays on a solid-support is essential for the realization of the DNA chip technology potential. In most DNA chip applications, the DNA arrays are used to capture or analyze the target sequences and/or detection probes via hybridization reactions alone (1-7) or with subsequent primer extension reactions (8). The reliability and integrity of the hybridization reactions are highly dependent, in addition to the actual base composition of the arrayed oligonucleotides, on the quality and the characteristics of the DNA arrays. In developing a useful and reliable chemistry for producing DNA arrays, the accessibility and functionality of the surface-bound DNA, the density of attachment, the stability of the array, the reproducibility of the attachment chemistry, and the fidelity of the immobilized sequences are all critical.There have been numerous reports regarding immobilization (9 -26) or direct synthesis (27, 28) of oligonucleotides on solid supports, such as glass, silicon, membranes, and polystyrene. Parallel synthesis of oligonucleotides directly onto the solid support by photoactivatable chemistries (27) or standard phosphoramidite chemistries (28) have, thus far, been the most successful approach to manufacturing high-density DNA arrays. Patterning of presynthesized oligonucleotides, however, is preferred for many research applications and low-to moderate-density-array applications requi...

Single nucleotide polymorphism (SNP) genotyping is playing an increasing role in genome mapping, pharmacogenetic studies, and drug discovery. To date, genome-wide scans and studies involving thousands of SNPs and samples have been hampered by the lack of a system that can perform genotyping with cost-effective throughput, accuracy, and reliability. To address this need, Orchid has developed an automated, ultra-high throughput system, SNPstream UHT, which uses multiplexed PCR in conjunction with our next generation SNP-ITtag array single base extension genotyping technology. The system employs oligonucleotide microarrays manufactured in a 384-well format on a novel glass-bottomed plate. Multiplexed PCR and genotyping are performed in homogeneous reactions, and assay results are read by direct two-color fluorescence on the SNPstream UHT Array Imager. The systems flexibility enables large projects involving thousands of SNPs and thousands of samples as well as small projects that have hundreds of SNPs and hundreds of samples to be done cost effectively. We have successfully demonstrated this system in greater than 1 000 000 genotyping assays with >96% of samples giving genotypes with >99% accuracy.

No abstract