Genome-wide detection of allelic imbalance using human SNPs and high-density DNA arrays.

Most human cancers are characterized by genomic instability, the accumulation of multiple genetic alterations and allelic imbalance throughout the genome. Loss of heterozygosity (LOH) is a common form of allelic imbalance and the detection of LOH has been used to identify genomic regions that harbor tumor suppressor genes and to characterize tumor stages and progression. Here we describe the use of high-density oligonucleotide arrays for genome-wide scans for LOH and allelic imbalance in human tumors. The arrays contain redundant sets of probes for 600 genetic loci that are distributed across all human chromosomes. The arrays were used to detect allelic imbalance in two types of human tumors, and a subset of the results was confirmed using conventional gel-based methods. We also tested the ability to study heterogeneous cell populations and found that allelic imbalance can be detected in the presence of a substantial background of normal cells. The detection of LOH and other chromosomal changes using large numbers of single nucleotide polymorphism (SNP) markers should enable identification of patterns of allelic imbalance with potential prognostic and diagnostic utility.

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.

Detection of heterozygous mutations in BRCA1 using high density oligonucleotide arrays and two-colour fluorescence analysis.

The ability to scan a large gene rapidly and accurately for all possible heterozygous mutations in large numbers of patient samples will be critical for the future of medicine. We have designed high-density arrays consisting of over 96,600 oligonucleotides 20-nucleotides (nt) in length to screen for a wide range of heterozygous mutations in the 3.45-kilobases (kb) exon 11 of the hereditary breast and ovarian cancer gene BRCA1. Reference and test samples were co-hybridized to these arrays and differences in hybridization patterns quantitated by two-colour analysis. Fourteen of fifteen patient samples with known mutations were accurately diagnosed, and no false positive mutations were identified in 20 control samples. Eight single nucleotide polymorphisms were also readily detected. DNA chip-based assays may provide a valuable new technology for high-throughput cost-efficient detection of genetic alterations.

Human cytomegalovirus encodes three G protein-coupled receptor homologues.

Human cytomegalovirus (HCMV) is a herpesvirus with a genome of 230 kilobases (Kb) encoding about 200 genes. Although infection is generally innocuous, HCMV causes serious congenital and neonatal disease, and is a dangerous opportunistic pathogen in immune-deficient individuals. We have identified a family of three HCMV genes which encode polypeptides containing seven putative membrane-spanning domains, and a series of well-defined motifs characteristic of the rhodopsin-like G protein-coupled receptors (GCRs). By these criteria all three of the HCMV sequences are homologous to cellular GCRs. Members of this receptor family function in visual signal transduction, regulation of homeostasis, and development, and include known and potential oncogenes. These receptors are activated by photons or small molecules such as neurotransmitters, and glycoprotein hormones. The finding of viral-encoded GCR homologues implies a further level of complexity in the interactions between HCMV and its host, and may provide a potential pathway for virally transformed cell proliferation. Their identification could permit the development of a novel class of antiviral drugs analogous to beta-adrenergic receptor antagonists.