A genotyping system capable of simultaneously analyzing >1000 single nucleotide polymorphisms in a haploid genome

A high-throughput genotyping system for scoring single nucleotide polymorphisms (SNPs) has been developed. With this system, >1000 SNPs can be analyzed in a single assay, with a sensitivity that allows the use of single haploid cells as starting material. In the multiplex polymorphic sequence amplification step, instead of attaching universal sequences to the amplicons, primers that are unlikely to have nonspecific and productive interactions are used. Genotypes of SNPs are then determined by using the widely accessible microarray technology and the simple single-base extension assay. Three SNP panels, each consisting of >1000 SNPs, were incorporated into this system. The system was used to analyze 24 human genomic DNA samples. With 5 ng of human genomic DNA, the average detection rate was 98.22% when single probes were used, and 96.71% could be detected by dual probes in different directions. When single sperm cells were used, 91.88% of the SNPs were detectable, which is comparable to the level that was reached when very few genetic markers were used. By using a dual-probe assay, the average genotyping accuracy was 99.96% for 5 ng of human genomic DNA and 99.95% for single sperm. This system may be used to significantly facilitate large-scale genetic analysis even if the amount of DNA template is very limited or even highly degraded as that obtained from paraffin-embedded cancer specimens, and to make many unpractical research projects highly realistic and affordable

Strong correlation between meiotic crossovers and haplotype structure in a 2.5-Mb region on the long arm of chromosome 21

Although the haplotype structure of the human genome has been studied in great detail, very little is known about the mechanisms underlying its formation. To investigate the role of meiotic recombination on haplotype block formation, single nucleotide polymorphisms were selected at a high density from a 2.5-Mb region of human chromosome 21. Direct analysis of meiotic recombination by high-throughput multiplex genotyping of 662 single sperm identifies 41 recombinants. The crossovers were nonrandomly distributed within 16 small areas. All, except one, of these crossovers fall in areas where the haplotype structure exhibits breakdown, displaying a strong statistically positive association between crossovers and haplotype block breaks. The data also indicate a particular clustered distribution of recombination hotspots within the region. This finding supports the hypothesis that meiotic recombination makes a primary contribution to haplotype block formation in the human genome

Discovery and Mechanistic Studies of Facile N‑Terminal Cα−C Bond Cleavages in the Dissociation of Tyrosine-Containing Peptide Radical Cations

Fascinating N-terminal Cα−C bond cleavages in a series of nonbasic tyrosine-containing peptide radical cations have been observed under low-energy collision-induced dissociation (CID), leading to the generation of rarely observed x-type radical fragments, with significant abundances. CID experiments of the radical cations of the alanyltyrosylglycine tripeptide and its analogues suggested that the N-terminal Cα−C bond cleavage, yielding its [x2 + H]•+ radical cation, does not involve an N-terminal α-carbon-centered radical. Theoretical examination of a prototypical radical cation of the alanyltyrosine dipeptide, using density functional theory calculations, suggested that direct N-terminal Cα−C bond cleavage could produce an ion−molecule complex formed between the incipient a1+ and x1• fragments. Subsequent proton transfer from the iminium nitrogen atom in a1+ to the acyl carbon atom in x1• results in the observable [x1 + H]•+. The barriers against this novel Cα−C bond cleavage and the competitive N−Cα bond cleavage, forming the complementary [c1 + 2H]+/[z1 − H]•+ ion pair, are similar (ca. 16 kcal mol−1). Rice−Ramsperger−Kassel−Marcus modeling revealed that [x1 + H]•+ and [c1 + 2H]+ species are formed with comparable rates, in agreement with energy-resolved CID experiments for [AY]•+