Fully Automated Protein Proximity Assay in Formalin-Fixed, Paraffin-Embedded Tissue Using Caged Haptens.

The ability to interrogate for the presence and distribution of protein-protein complexes (PPCs) is of high importance for the advancement of diagnostic capabilities such as determining therapeutic effects in the context of pharmaceutical development. Herein, we report a novel assay for detecting and visualizing PPCs on formalin-fixed, paraffin-embedded material using a caged hapten. To this end, we synthetically modified a nitropyrazole hapten with an alkaline phosphatase (AP)-responsive self-immolative caging group. The AP-labile caging group abrogates antibody binding; however, upon exposure to AP, the native hapten is regenerated. These caged haptens were applied in a proximity assay format by the use of a first antibody labeled with caged haptens that can be uncaged by AP conjugated to the second antibody. Only when the two epitopes of interest are in close proximity to one another will the AP interact with the caged hapten and uncage it. The native hapten, which represents the population of PPCs, was then visualized by an anti-hapten antibody conjugated to horseradish peroxidase, followed by diaminobenzidine detection. We provide proof of concept for the detection of protein proximity pairs (ß-catenin-E-cadherin and EGFR-GRB2), and confirm assay specificity through technical controls involving reagent omission experiments, and biologically by treatment with small-molecule kinase inhibitors that interrupt kinase-adaptor complexes.

Organism identification using a genome sequence-independent universal microarray probe set.

There has been increasing interest and efforts devoted to developing biosensor technologies for identifying pathogens, particularly in the biothreat area. In this study, a universal set of short 12- and 13-mer oligonucleotide probes was derived independently of a priori genomic sequence information and used to generate unique species-dependent genomic hybridization signatures. The probe set sequences were algorithmically generated to be maximally distant in sequence space and not dependent on the sequence of any particular genome. The probe set is universally applicable because it is unbiased and independent of hybridization predictions based upon simplified assumptions regarding probe-target duplex formation from linear sequence analysis. Tests were conducted on microarrays containing 14,283 unique probes synthesized using an in situ light-directed synthesis methodology. The genomic DNA hybridization intensity patterns reproducibly differentiated various organisms (Bacillus subtilis, Yersinia pestis, Streptococcus pneumonia, Bacillus anthracis, and Homo sapiens), including the correct identification of a blinded "unknown" sample. Applications of this method include not only pathological and forensic genome identification in medicine and basic science, but also potentially a novel method for the discovery of unknown targets and associations inherent in dynamic nucleic acid populations such as represented by differential gene expression.

How independent are the appearances of n-mers in different genomes?

MOTIVATION: Analysis of statistical properties of DNA sequences is important for evolutional biology as well as for DNA probe and PCR technologies. These technologies, in turn, can be used for organism identification, which implies applications in the diagnosis of infectious diseases, environmental studies, etc. RESULTS: We present results of the correlation analysis of distributions of the presence/absence of short nucleotide subsequences of different length ('n-mers', n = 5-20) in more than 1500 microbial and virus genomes, together with five genomes of multicellular organisms (including human). We calculate whether a given n-mer is present or absent (frequency of presence) in a given genome, which is not the usually calculated number of appearances of n-mers in one or more genomes (frequency of appearance). For organisms that are not close relatives of each other, the presence/absence of different 7-20mers in their genomes are not correlated. For close biological relatives, some correlation of the presence of n-mers in this range appears, but is not as strong as expected. Suppressed correlations among the n-mers present in different genomes leads to the possibility of using random sets of n-mers (with appropriately chosen n) to discriminate genomes of different organisms and possibly individual genomes of the same species including human with a low probability of error.

A scalable high-throughput chemical synthesizer.

A machine that employs a novel reagent delivery technique for biomolecular synthesis has been developed. This machine separates the addressing of individual synthesis sites from the actual process of reagent delivery by using masks placed over the sites. Because of this separation, this machine is both cost-effective and scalable, and thus the time required to synthesize 384 or 1536 unique biomolecules is very nearly the same. Importantly, the mask design allows scaling of the number of synthesis sites without the addition of new valving. Physical and biological comparisons between DNA made on a commercially available synthesizer and this unit show that it produces DNA of similar quality.