We have improved the "polymerase chain reaction" (PCR) to permit rapid analysis of any known mutation in genomic DNA. We demonstrate a system, ARMS (Amplification Refractory Mutation System), that allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis. The system is simple, reliable and non-isotopic. It will clearly distinguish heterozygotes at a locus from homozygotes for either allele. The system requires neither restriction enzyme digestion, allele-specific oligonucleotides as conventionally applied, nor the sequence analysis of PCR products. The basis of the invention is that unexpectedly, oligonucleotides with a mismatched 3'-residue will not function as primers in the PCR under appropriate conditions. We have analysed DNA from patients with alpha 1-antitrypsin (AAT) deficiency, from carriers of the disease and from normal individuals. Our findings are in complete agreement with allele assignments derived by direct sequencing of PCR products.

The recent development of yeast artificial chromosome (YAC) vectors has provided a system for cloning fragments that are over ten times larger than those that can be cloned in more established systems. We have developed a method for the rapid isolation of terminal sequences from YAC clones. The YAC clone is digested with a range of restriction enzymes, a common linker is ligated to the DNA fragments and terminal sequences are amplified using a vector specific primer and a linker specific primer. Sequence data derived from these terminal specific products can be used to design primers for a further round of screening to isolate overlapping clones. The method also provides a convenient method of generating Sequence Tagged Sites for the mapping of complex genomes.

In the arterial circulation, regions of disturbed flow (DF), which are characterized by flow separation and transient vortices, are susceptible to atherogenesis, whereas regions of undisturbed laminar flow (UF) appear protected. Coordinated regulation of gene expression by endothelial cells (EC) may result in differing regional phenotypes that either favor or inhibit atherogenesis. Linearly amplified RNA from freshly isolated EC of DF (inner aortic arch) and UF (descending thoracic aorta) regions of normal adult pigs was used to profile differential gene expression reflecting the steady state in vivo. By using human cDNA arrays, Ϸ2,000 putatively differentially expressed genes were identified through false-discovery-rate statistical methods. A sampling of these genes was validated by quantitative realtime PCR and͞or immunostaining en face. Biological pathway analysis revealed that in DF there was up-regulation of several broad-acting inflammatory cytokines and receptors, in addition to elements of the NF-B system, which is consistent with a proinflammatory phenotype. However, the NF-B complex was predominantly cytoplasmic (inactive) in both regions, and no significant differences were observed in the expression of key adhesion molecules for inflammatory cells associated with early atherogenesis. Furthermore, there was no histological evidence of inflammation. Protective profiles were observed in DF regions, notably an enhanced antioxidative gene expression. This study provides a public database of regional EC gene expression in a normal animal, implicates hemodynamics as a contributory mechanism to athero-susceptibility, and reveals the coexistence of pro-and antiatherosclerotic transcript profiles in susceptible regions. The introduction of additional risk factors may shift this balance to favor lesion development.

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We show here that UV absorbance of denatured adeno-associated virus (AAV) vector provides a simple, rapid, and direct method for quantifying vector genomes and capsid proteins in solution. We determined the molar extinction coefficients of capsid protein to be 3.72 x 10(6) M(-1) cm(-1) at 260 nm and 6.61 x 10(6) M(-1) cm(-1) at 280 nm. For recombinant AAV vectors, extinction coefficients can be calculated by including the predicted absorbance of the vector DNA. Since the amount of empty capsids in purified vector preparations lowers the A(260)/A(280) ratio in a predictable manner, the vector genome (vg) and capsid particle (cp) titers in purified AAV vector preparations can be calculated from the absorbance at 260 nm and the A(260)/A(280) ratio. To validate this method, the vg and cp titers calculated by UV absorbance were compared with titers determined by quantitative (Q)-PCR and capsid ELISA. The vg titers determined by absorbance agreed well with titers determined by Q-PCR. The cp/vg ratio determined by the A(260)/A(280) method also correlated well with those determined by AAV capsid ELISA in conjunction with Q-PCR. This new method provides a simple and rapid means to determine AAV vg titers and the ratio of empty to full particles in purified virus preparations.

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