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.

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.

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.

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.