High density synthetic oligonucleotide arrays.

Experimental genomics involves taking advantage of sequence information to investigate and understand the workings of genes, cells and organisms. We have developed an approach in which sequence information is used directly to design high-density, two-dimensional rays of synthetic oligonucleotides. The GeneChipe probe arrays are made using spatially patterned, light-directed combinatorial chemical synthesis and contain up to hundreds of thousands of different oligonucleotides on a small glass surface. The arrays have been designed and used for quantitative and highly parallel measurements of gene expression, to discover polymorphic loci and to detect the presence of thousands of alternative alleles. Here, we describe the fabrication of the arrays, their design and some specific applications to high-throughput genetic and cellular analysis.

Patterns of single-nucleotide polymorphisms in candidate genes for blood-pressure homeostasis.

Sequence variation in human genes is largely confined to single-nucleotide polymorphisms (SNPs) and is valuable in tests of association with common diseases and pharmacogenetic traits. We performed a systematic and comprehensive survey of molecular variation to assess the nature, pattern and frequency of SNPs in 75 candidate human genes for blood-pressure homeostasis and hypertension. We assayed 28 Mb (190 kb in 148 alleles) of genomic sequence, comprising the 5' and 3' untranslated regions (UTRs), introns and coding sequence of these genes, for sequence differences in individuals of African and Northern European descent using high-density variant detection arrays (VDAs). We identified 874 candidate human SNPs, of which 22% were confirmed by DNA sequencing to reveal a discordancy rate of 21% for VDA detection. The SNPs detected have an average minor allele frequency of 11%, and 387 are within the coding sequence (cSNPs). Of all cSNPs, 54% lead to a predicted change in the protein sequence, implying a high level of human protein diversity. These protein-altering SNPs are 38% of the total number of such SNPs expected, are more likely to be population-specific and are rarer in the human population, directly demonstrating the effects of natural selection on human genes. Overall, the degree of nucleotide polymorphism across these human genes, and orthologous great ape sequences, is highly variable and is correlated with the effects of functional conservation on gene sequences.

Determination of ancestral alleles for human single-nucleotide polymorphisms using high-density oligonucleotide arrays.

Here we report the application of high-density oligonucleotide array (DNA chip)-based analysis to determine the distant history of single nucleotide polymorphisms (SNPs) in current human populations. We analysed orthologues for 397 human SNP sites (identified in CEPH pedigrees from Amish, Venezuelan and Utah populations) from 23 common chimpanzee, 19 pygmy chimpanzee and 11 gorilla genomic DNA samples. From this data we determined 214 proposed ancestral alleles (the sequence found in the last common ancestor of humans and chimpanzees). In a diverse human population set, we found that SNP alleles with higher frequencies were more likely to be ancestral than less frequently occurring alleles. There were, however, exceptions. We also found three shared human/pygmy chimpanzee polymorphisms, all involving CpG dinucleotides, and two shared human/gorilla polymorphisms, one involving a CpG dinucleotide. We demonstrate that microarray-based assays allow rapid comparative sequence analysis of intra- and interspecies genetic variation.

Characterization of single-nucleotide polymorphisms in coding regions of human genes.

A major goal in human genetics is to understand the role of common genetic variants in susceptibility to common diseases. This will require characterizing the nature of gene variation in human populations, assembling an extensive catalogue of single-nucleotide polymorphisms (SNPs) in candidate genes and performing association studies for particular diseases. At present, our knowledge of human gene variation remains rudimentary. Here we describe a systematic survey of SNPs in the coding regions of human genes. We identified SNPs in 106 genes relevant to cardiovascular disease, endocrinology and neuropsychiatry by screening an average of 114 independent alleles using 2 independent screening methods. To ensure high accuracy, all reported SNPs were confirmed by DNA sequencing. We identified 560 SNPs, including 392 coding-region SNPs (cSNPs) divided roughly equally between those causing synonymous and non-synonymous changes. We observed different rates of polymorphism among classes of sites within genes (non-coding, degenerate and non-degenerate) as well as between genes. The cSNPs most likely to influence disease, those that alter the amino acid sequence of the encoded protein, are found at a lower rate and with lower allele frequencies than silent substitutions. This likely reflects selection acting against deleterious alleles during human evolution. The lower allele frequency of missense cSNPs has implications for the compilation of a comprehensive catalogue, as well as for the subsequent application to disease association.

Genome-wide mapping with biallelic markers in Arabidopsis thaliana.

Single-nucleotide polymorphisms, as well as small insertions and deletions (here referred to collectively as simple nucleotide polymorphisms, or SNPs), comprise the largest set of sequence variants in most organisms. Positional cloning based on SNPs may accelerate the identification of human disease traits and a range of biologically informative mutations. The recent application of high-density oligonucleotide arrays to allele identification has made it feasible to genotype thousands of biallelic SNPs in a single experiment. It has yet to be established, however, whether SNP detection using oligonucleotide arrays can be used to accelerate the mapping of traits in diploid genomes. The cruciferous weed Arabidopsis thaliana is an attractive model system for the construction and use of biallelic SNP maps. Although important biological processes ranging from fertilization and cell fate determination to disease resistance have been modelled in A. thaliana, identifying mutations in this organism has been impeded by the lack of a high-density genetic map consisting of easily genotyped DNA markers. We report here the construction of a biallelic genetic map in A. thaliana with a resolution of 3.5 cM and its use in mapping Eds16, a gene involved in the defence response to the fungal pathogen Erysiphe orontii. Mapping of this trait involved the high-throughput generation of meiotic maps of F2 individuals using high-density oligonucleotide probe array-based genotyping. We developed a software package called InterMap and used it to automatically delimit Eds16 to a 7-cM interval on chromosome 1. These results are the first demonstration of biallelic mapping in diploid genomes and establish means for generalizing SNP-based maps to virtually any genetic organism.

Large-scale identification, mapping, and genotyping of single-nucleotide polymorphisms in the human genome.

Single-nucleotide polymorphisms (SNPs) are the most frequent type of variation in the human genome, and they provide powerful tools for a variety of medical genetic studies. In a large-scale survey for SNPs, 2.3 megabases of human genomic DNA was examined by a combination of gel-based sequencing and high-density variation-detection DNA chips. A total of 3241 candidate SNPs were identified. A genetic map was constructed showing the location of 2227 of these SNPs. Prototype genotyping chips were developed that allow simultaneous genotyping of 500 SNPs. The results provide a characterization of human diversity at the nucleotide level and demonstrate the feasibility of large-scale identification of human SNPs.

Using oligonucleotide probe arrays to access genetic diversity.

As the Human Genome Project and related efforts identify and determine the DNA sequences of human genes, it is important that highly reliable and efficient mechanisms are found to access individual genetic variation. It is only through a greater understanding of genetic diversity that the true benefit of the Human Genome Project will be realized. One approach, hybridization to high-density arrays of oligonucleotides, is a fast and effective means of accessing this genetic variation. Light-directed chemical synthesis has been used to generate miniaturized, high-density arrays of oligonucleotide probes. Application-specific oligonucleotide probe array designs have been developed for the rapid screening of characterized genes. Dedicated instrumentation and software have been developed for array hybridization, fluorescence detection and data acquisition and analysis. In a specific and challenging application, oligonucleotide probe arrays have been used to screen the reverse transcriptase and protease genes of the highly polymorphic HIV-1 genome to explore genetic diversity and detect mutations conferring resistance to antiviral drugs. Results from this application strongly suggest that oligonucleotide probe arrays will be a powerful tool for rapid investigations in sequence checking, pathogen detection, expression monitoring and DNA molecular recognition.

Likelihood DNA sequencing by hybridization.

Sequencing by hybridization (SBH) extracts local sequence information from a DNA fragment using hybridization with oligonucleotides and then reconstructs the sequence using the derived information. We describe an improvement to the SBH methodology which will allow it to work efficiently in the presence of hybridization errors. In particular, given a set of hybridizing probes, and the empirically derived rates of false positive and false negative hybridization, we can estimate the most likely DNA fragment to have produced the set of probes, and then estimate the probability that it generated the hybridization data. This methodology extends earlier results by identifying the most probable fragment to have generated the actual hybridization data. The methodology described will also generate longer unambiguous sequence fragments without the use of overlapping fragments.

Mapping genomic library clones using oligonucleotide arrays.

We have developed a high-density DNA probe array and accompanying biochemical and informatic methods to order clones from genomic libraries. This approach involves a series of enzymatic steps for capturing a set of short dispersed sequence markers scattered throughout a high-molecular-weight DNA. By this process, all the ambiguous sequences lying adjacent to a given Type IIS restriction site are ligated between two DNA adapters. These markers, once amplified and labeled by PCR, can be hybridized and detected on a high-density oligonucleotide array bearing probes complementary to all possible markers. The array is synthesized using light-directed combinatorial chemistry. For each clone in a genomic library, a characteristic set of sequence markers can be determined. On the basis of the similarity between the marker sets for each pair of clones, their relative overlap can be measured. The library can be sequentially ordered into a contig map using this overlap information. This new methodology does not require gel-based methods or prior sequence information and involves manipulations that should allow for easy adaptation to automated processing and data collection.

DNA sequence confidence estimation.

A significant bottleneck in the current DNA sequencing process is the manual editing of trace data generated by automated DNA sequencers. This step is used to correct base calls and to associate to each base call a confidence level. The confidence levels are used in the assembly process to determine overlaps and to resolve discrepancies in determining the consensus sequence. This single step may cost as much as 4 to 8 cents per finished base. We report an approach to automated trace editing using classification trees to detect and exploit context-based patterns in trace peak heights. Local base composition and nearby peak heights account for 80% of the variations in peak heights. Classification algorithms were developed to identify 37% of automated base calls that differ from the consensus sequence. With these algorithms, 12% of the base calls had confidence levels less than 90%.

Mutation detection by ligation to complete n-mer DNA arrays.

A new approach to comparative nucleic acid sequence analysis is described that uses the ligation of DNA targets to high-density arrays containing complete sets of covalently attached oligonucleotides of length eight and nine. The combination of enzymatic or chemical ligation with a directed comparative analysis avoids many of the intrinsic difficulties associated with hybridization-based de novo sequence reconstruction methods described previously. Double-stranded DNA targets were fragmented and labeled to produce quasirandom populations of 5' termini suitable for ligation and detection on the arrays. Kilobase-size DNA targets were used to demonstrate that complete n-mer arrays can correctly verify known sequences and can determine the presence of sequence differences relative to a reference. By use of 9-mer arrays, sequences of 1.2-kb targets were verified with >99.9% accuracy. Mutations in target sequences were detected by directly comparing the intensity pattern obtained for an unknown with that obtained for a known reference sequence. For targets of moderate length (1.2 kb), 100% of the mutations in the queried sequences were detected with 9-mer arrays. For higher complexity targets (2.5 and 16.6 kb), a relatively high percentage of mutations (90% and 66%, respectively) were correctly identified with a low false-positive rate of <0.03 percent. The methods described provide a general approach to analyzing nucleic acid samples on the basis of the interpretation of sequence-specific patterns of hybridization and ligation on complete n-mer oligonucleotide arrays.

Affinity panning of a library of peptides displayed on bacteriophages reveals the binding specificity of BiP.

We have used affinity panning of libraries of bacteriophages that display random octapeptide or dodecapeptide sequences at the N-terminus of the adsorption protein (pIII) to characterize peptides that bind to the endoplasmic reticulum chaperone BiP and to develop a scoring system that predicts potential BiP-binding sequences in naturally occurring polypeptides. BiP preferentially binds peptides containing a subset of aromatic and hydrophobic amino acids in alternating positions, suggesting that peptides bind in an extended conformation, with the side chains of alternating residues pointing into a cleft on the BiP molecule. Synthetic peptides with sequences corresponding to those displayed by BiP-binding bacteriophages bind to BiP and stimulate its ATPase activity, with a half-maximal concentration in the range 10-60 microM.

Parallel genotyping of human SNPs using generic high-density oligonucleotide tag arrays.

Large scale human genetic studies require technologies for generating millions of genotypes with relative ease but also at a reasonable cost and with high accuracy. We describe a highly parallel method for genotyping single nucleotide polymorphisms (SNPs), using generic high-density oligonucleotide arrays that contain thousands of preselected 20-mer oligonucleotide tags. First, marker-specific primers are used in PCR amplifications of genomic regions containing SNPs. Second, the amplification products are used as templates in single base extension (SBE) reactions using chimeric primers with 3' complementarity to the specific SNP loci and 5' complementarity to specific probes, or tags, synthesized on the array. The SBE primers, terminating one base before the polymorphic site, are extended in the presence of labeled dideoxy NTPs, using a different label for each of the two SNP alleles, and hybridized to the tag array. Third, genotypes are deduced from the fluorescence intensity ratio of the two colors. This approach takes advantage of multiplexed sample preparation, hybridization, and analysis at each stage. We illustrate and test this method by genotyping 44 individuals for 142 human SNPs identified previously in 62 candidate hypertension genes. Because the hybridization results are quantitative, this method can also be used for allele-frequency estimation in pooled DNA samples.