High-density oligonucleotide arrays can be used to rapidly examine large amounts of DNA sequence in a high throughput manner. An array designed to determine the specific nucleotide sequence of 705 bp of the rpoB gene of Mycobacterium tuberculosis accurately detected rifampin resistance associated with mutations of 44 clinical isolates of M. tuberculosis. The nucleotide sequence diversity in 121 Mycobacterial isolates (comprised of 10 species) was examined by both conventional dideoxynucleotide sequencing of the rpoB and 16S genes and by analysis of the rpoB oligonucleotide array hybridization patterns. Species identification for each of the isolates was similar irrespective of whether 16S sequence, rpoB sequence, or the pattern of rpoB hybridization was used. However, for several species, the number of alleles in the 16S and rpoB gene sequences provided discordant estimates of the genetic diversity within a species. In addition to confirming the array's intended utility for sequencing the region of M. tuberculosis that confers rifampin resistance, this work demonstrates that this array can identify the species of nontuberculous Mycobacteria. This demonstrates the general point that DNA microarrays that sequence important genomic regions (such as drug resistance or pathogenicity islands) can simultaneously identify species and provide some insight into the organism's population structure.[The sequence data described in this paper have been submitted to GenBank under accession nos. AF09766-AF059853 and AF060279-AF060367.]For patients infected with Mycobacteria, especially those coinfected with the human immunodeficiency virus type 1 and type 2 (HIV-1, HIV-2), the identity of the Mycobacterium species and the presence of mutations that confer both biologically and clinically important phenotypes are of critical importance. Both of these issues have implications for the appropriate care and treatment of the infected patient. For example, although M. avium complex (MAC) is the most common cause for both disseminated Mycobacterium disease and death in patients with AIDS in the developed world (∼25%-50% of adults and 10% of children with AIDS are infected [Inderlied et al. 1993], Mycobacterium tuberculosis infections are also found in these patient populations. Important public health and patient management decisions (e.g., the need for clinical isolation and the choice of the appropriate therapeutic regimen) depend on a timely and accurate identification of the infecting agent. (Moore et al. 1997).On the basis of the insights provided by previously characterized RIF resistant mutants in Escherichia coli (Ovchinnikov et al. 1983; Jin and Gross 7 Corresponding author. E-MAIL tom gingeras@affymetrix.com; FAX (408) 481-0422.

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A method for identifying and validating single nucleotide polymorphisms (SNPs) with high-density oligonucleotide arrays without the need for locus-specific polymerase chain reactions (PCR) is described in this report. Genomic DNAs were divided into subsets with complexity of ∼10 Mb by restriction enzyme digestion and gel-based fragment size resolution, ligated to a common adaptor, and amplified with one primer in a single PCR reaction. As a demonstration of this approach, a total of 124 SNPs were located in 190 kb of genomic sequences distributed across the entire human genome by hybridizing to high-density variant detection arrays (VDA). A set of independent validation experiments was conducted for these SNPs employing bead-based affinity selection followed by hybridization of the affinity-selected SNP-containing fragments to the same VDA that was used to identify the SNPs. A total of 98.7% (74/75) of these SNPs were confirmed using both DNA dideoxynucleotide sequencing and the VDA methodologies. With flexible sample preparation, high-density oligonucleotide arrays can be tailored for even larger scale genome-wide SNP discovery as well as validation.With the completion of the first draft of human genomic consensus sequence, there is a predicable increase in interest concerning the types and amount of genetic diversity in the human population. The most abundant type of variations are single nucleotide polymorphisms (SNPs) (Collins et al. 1997). Some of these genetic variations underpin the genetic basis for disease susceptibility and are used in genetic studies as markers for complex traits and diseases.High-density variation detection arrays (VDAs) have been successfully used for large-scale SNP screening (Chee et al. 1996;Wang et al. 1998;Cargill et al. 1999;Hacia et al. 1999;Halushka et al. 1999). In these experiments, predetermined DNA targets within the genome were amplified with specific PCR reactions. To discover SNPs in this way, each locus in the genome needs to be amplified individually. Although multiple loci can be amplified in a multiplex PCR, this multiplexing approach is costly and labor intensive in the development stages and difficult to scale.In this study, high-density oligonucleotide arrays were used to screen for SNPs from a subset of human genomic DNA. Each subset of genomic DNAs contained a complexity up to 10 Mb and was amplified with one primer in a single PCR reaction. Sequence variations were screened with the same efficiency as individually amplified targets by hybridizing the same complex genomic sample to multiple designs of VDA. In addition, the SNP candidates were validated by using dideoxynucleotide sequencing and by using a bead-based affinity method to enrich fragments that contained candidate SNPs followed by reanalyzing these enriched fragments in a second round of VDA analysis.

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The corpus callosum has been implicated as a region of dysfunctional connectivity in schizophrenia, but the association between age and callosal pathology is unclear. Magnetic resonance imaging (MRI) and diffusion-tensor imaging (DTI) were performed on adults (n=34) and adolescents (n=17) with schizophrenia and adult (n=33) and adolescent (n=15) age- and sex-matched healthy controls. The corpus callosum was manually traced on each participant’s MRI, and the DTI scan was co-registered to the MRI. The corpus callosum was divided into five anteroposterior segments. Area and anisotropy were calculated for each segment. Both patient groups demonstrated reduced callosal anisotropy; however, the adolescents exhibited reductions mostly in anterior regions while the reductions were more prominent in posterior regions of the adults. The adolescent patients showed greater decreases in absolute area as compared with the adult patients, particularly in the anterior segments. However, the adults showed greater reductions when area was considered relative to whole brain white matter volume. Our results suggest that the initial stages of the illness are characterized by deficiencies in frontal connections, and the chronic phase is characterized by deficits in the posterior corpus callosum; or, alternatively, adolescent-onset schizophrenia may represent a different or more severe form of the illness.

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