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.

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

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.

Two color hybridization analysis using high density oligonucleotide arrays and energy transfer dyes.

High density oligonucleotide arrays (DNA chips) have been used in two color mutational analysis of the 3.43 kb exon 11 of the hereditary breast and ovarian cancer gene BRCA1 . Two color analysis allows competitive hybridization between a reference standard and an unknown sample, improving the performance of the assay. Fluorescein and phycoerythrin dyes werepreviously used due to their compatibility with a single line 488 nm excitation source. Here we show that an alternative dye combination, containing the energy transfer dye system phycoerythrin*cy5 along with phycoerythrin, provides more evenly matched signal intensities and decreased spectral overlap between the two fluorophores, while maintaining compatibility with a 488 nm excitation source.

Evolutionary sequence comparisons using high-density oligonucleotide arrays.

We explored the utility of high-density oligonucleotide arrays (DNA chips) for obtaining sequence information from homologous genes in closely related species. Orthologues of the human BRCA1 exon 11, all approximately 3.4 kb in length and ranging from 98.2% to 83.5% nucleotide identity, were subjected to hybridization-based and conventional dideoxysequencing analysis. Retrospective guidelines for identifying high-fidelity hybridization-based sequence calls were formulated based upon dideoxysequencing results. Prospective application of these rules yielded base-calling with at least 98.8% accuracy over orthologous sequence tracts shown to have approximately 99% identity. For higher primate sequences with greater than 97% nucleotide identity, base-calling was made with at least 99.91% accuracy covering a minimum of 97% of the sequence. Using a second-tier confirmatory hybridization chip strategy, shown in several cases to confirm the identity of predicted sequence changes, the complete sequence of the chimpanzee, gorilla and orangutan orthologues should be deducible solely through hybridization-based methodologies. Analysis of less highly conserved orthologues can still identify conserved nucleotide tracts of at least 15 nucleotides and can provide useful information for designing primers. DNA-chip based assays can be a valuable new technology for obtaining high-throughput cost-effective sequence information from related genomes.

Strategies for mutational analysis of the large multiexon ATM gene using high-density oligonucleotide arrays.

Mutational analysis of large genes with complex genomic structures plays an important role in medical genetics. Technical limitations associated with current mutation screening protocols have placed increased emphasis on the development of new technologies to simplify these procedures. High-density arrays of >90,000-oligonucleotide probes, 25 nucleotides in length, were designed to screen for all possible heterozygous germ-line mutations in the 9.17-kb coding region of the ATM gene. A strategy for rapidly developing multiexon PCR amplification protocols in DNA chip-based hybridization analysis was devised and implemented in preparing target for the 62 ATM coding exons. Improved algorithms for interpreting data from two-color experiments, where reference and test samples are cohybridized to the arrays, were developed. In a blinded study, 17 of 18 distinct heterozygous and 8 of 8 distinct homozygous sequence variants in the assayed region were detected accurately along with five false-positive calls while scanning >200 kb in 22 genomic DNA samples. Of eight heterozygous sequence changes found in more than one sample, six were detected in all cases. Five previously unreported sequence changes, not found by other mutational scanning methodologies on these same samples, were detected that led to either amino acid changes or premature truncation of the ATM protein. DNA chip-based assays should play a valuable role in high throughput sequence analysis of complex genes.

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.

Combinatorial chemistry--applications of light-directed chemical synthesis.

Combinatorial methods in biology and chemistry are proving to be powerful methods for generating molecular diversity. One approach, light-directed chemical synthesis, combines semiconductor-based photolithography technologies with solid-phase organic chemistry to synthesize large arrays of molecules with potential biological activity. This novel technology has the potential to provide libraries of both natural and synthetic molecules that might be screened rapidly for biological activity.

Multiplexed biochemical assays with biological chips.

High density peptide and oligonucleotide chips are fabricated using semiconductor-based technologies. These chips have a variety of biological applications.

Comparison of lipid/gramicidin dispersions and cocrystals by Raman scattering.

Gramicidin crystals, dimyristoylphosphatidylcholine (DMPC)/gramicidin dispersions, and DMPC/gramicidin cocrystals were examined by Raman scattering to determine lipid/gramicidin stoichiometries and lipid organization. Calibrations of the choline (716-cm-1) and tryptophan (756-cm-1) peaks indicate that the cocrystals contain two lipids for each gramicidin monomer, a result confirmed by chemical analyses of washed crystals. In dispersions with high lipid/gramicidin ratios (e.g., 25:1), the lipid is ordered but becomes increasingly disordered as the gramicidin content is increased. Paradoxically, the DMPC/gramicidin cocrystals have highly ordered lipids that possibly contain no gauche bonds at all, despite their low lipid/gramicidin ratio. In addition, the polypeptide amide I peak position near 1670 cm-1 is found to be independent of the lipid/gramicidin ratio in the complexes and may indicate a beta-helix-type secondary structure at all ratios. However, the amide I peak broadens significantly at low lipid/gramicidin ratios and broadens still further in the cocrystals, suggesting that protein-protein interactions may induce band-broadening distortions of the polypeptide structure.