Single nucleotide polymorphism (SNP) genotyping is playing an increasing role in genome mapping, pharmacogenetic studies, and drug discovery. To date, genome-wide scans and studies involving thousands of SNPs and samples have been hampered by the lack of a system that can perform genotyping with cost-effective throughput, accuracy, and reliability. To address this need, Orchid has developed an automated, ultra-high throughput system, SNPstream UHT, which uses multiplexed PCR in conjunction with our next generation SNP-ITtag array single base extension genotyping technology. The system employs oligonucleotide microarrays manufactured in a 384-well format on a novel glass-bottomed plate. Multiplexed PCR and genotyping are performed in homogeneous reactions, and assay results are read by direct two-color fluorescence on the SNPstream UHT Array Imager. The systems flexibility enables large projects involving thousands of SNPs and thousands of samples as well as small projects that have hundreds of SNPs and hundreds of samples to be done cost effectively. We have successfully demonstrated this system in greater than 1 000 000 genotyping assays with >96% of samples giving genotypes with >99% accuracy.

1,4-Dimethylbenzo[c]phenanthrene (1,4-DMBcPh) is the dimethylated analogue of the benzo[c]phenanthrene (BcPh), where one of its two methyl groups resides in the highly congested fjord-region. A comparative X-ray crystallographic analysis, described herein, shows that BcPh is distorted out-of-plane so that there is an angle of 27° between the outermost rings. The additional methyl groups in 1,4-DMBcPh increase this nonplanarity to an angle of 37°. This methyl group-induced disruption of planarity results in P and M enantiomers of 1,4-DMBcPh, and this helicity is observed in a pronounced manner in its putative metabolites, the dihydrodiol and diol epoxide. Synthetically, photochemical cyclization offers convenient access to 1,4-DMBcPh as well as its metabolites. For this, Wittig reaction of 2,5-dimethylbenzyltriphenylphosphonium chloride and 2-naphthaldehyde provided a cis/trans mixture of alkenes which, when subjected to photolysis, provided 1,4-DMBcPh. Despite the high steric congestion in the fjord-region, this reaction proceeds with respectable yields. Correspondingly, a Wittig reaction of the same phosphonium chloride derivative with 6-methoxy-2-naphthaldehyde afforded an alkene mixture that, upon photochemical ring closure, provided 10-methoxy-1,4-dimethylbenzo[c]phenanthrene. The methoxy group was cleaved and the resulting phenol oxidized to the o-quinone. Reduction of the o-quinone with NaBH4 under an oxygen atmosphere provided the dihydrodiol, (±)-trans-9,10-dihydroxy-9,10-dihydro-1,4-dimethylbenzo[c]phenanthrene. This dihydrodiol was found to be an approximately 3:1 mixture of diastereomers, which was produced as follows. Reduction of the quinone proceeds in a stereoselective manner producing both the (R,R)- and (S,S)-trans-diols. However, this factor, when coupled with the P, M atropisomerism of the hydrocarbon, results in two diastereomeric pairs of enantiomeric dihydrodiols. Due to steric constraints imposed by the fjord-region methyl group, the P → M interconversion is slow, making the proton resonances of the diastereomeric dihydrodiols distinctly observable by NMR. Assignment of major and minor dihydrodiol isomers has been achieved by NOESY experiments. Finally, epoxidation provides a mixture of diol epoxides that reflects the dihydrodiol ratio. The metabolic activation of these compounds to reactive intermediates was studied through analysis of their binding to DNA. DNA binding data using human mammary carcinoma MCF-7 cells reveal that the level of DNA binding of BcPh is not statistically different from that of 1,4-DMBcPh. However, there is an 11-fold increased activation of BcPh dihydrodiol as compared to the 1,4-DMBcPh dihydrodiol. In contrast to the planar benzo[a]pyrene, BcPh is only poorly adducted to DNA in culture cells. Thus, it appears that increasing the nonplanarity in this type of PAH lowers its ability to be metabolically activated to form DNA-damaging adducts, although in the case of 1,4-DMBcPh, the presence of the two methyl groups in one of the angular rings may also contribute to t...

A new approach to increase the selectivity of interaction between oligonucleotide probes and target nucleic acids is described. In place of a single, relatively long oligonucleotide probe, two or three short oligomers terminated by thiophosphoryl and bromoacetamido groups are employed. Fast and efficient autoligation takes place when the oligomers hybridize in a contiguous mode to the same complementary strand such that a thiophosphoryl group on one strand and a bromoacetamido group on another are brought into proximity. A single nucleotide mismatch for the short probes leads to marked reduction in the rate of autoligation. The binding affinity of the product is close to that for a natural probe of the same length. This approach could have potential in oligonucleotide-based diagnostics, chemical amplification systems, and therapeutic applications.