Search PubMed⌕ Search
Home/Archive/PubMed

PubMed · 11689121

A versatile high throughput screen for dioxygenase activity using solid-phase digital imaging.

J M Joern·T Sakamoto·A Arisawa·F H Arnold
View original source ↗Export metadata ↓

Abstract

We have developed a solid-phase, high throughput (10,000 clones/day) screen for dioxygenase activity. The cis-dihydrodiol product of dioxygenase bioconversion is converted to a phenol by acidification or to a catechol by reaction with cis-dihydrodiol dehydrogenase. Gibbs reagent reacts quickly with these oxygenated aromatics to yield colored products that are quantifiable using a microplate reader or by digital imaging and image analysis. The method is reproducible and quantitative at product concentrations of only 30 microM, with essentially no background from media components. This method is an effective general screen for aromatic oxidation and should be a useful tool for the discovery and directed evolution of oxygenases.

Explore related subjects

AutomationChlorobenzenesDrug Evaluation, PreclinicalImage Processing, Computer-AssistedModels, ChemicalOxidoreductasesOxygenOxygenasesPolymerase Chain ReactionProtein BindingJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, Non-P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

J M Joern, T Sakamoto, A Arisawa, F H Arnold. 2001. A versatile high throughput screen for dioxygenase activity using solid-phase digital imaging.. https://doi.org/10.1177/108705710100600403

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed2002

Direct detection of isotopically labeled metabolites bound to a protein microarray using a charge-coupled device.

A charge-coupled device (CCD) was used to quantitatively detect isotope-labeled ligands bound to a protein microarray. Protein microarrays with protein dots, 10-50 microm in diameter, were fabricated on an aluminized Mylar film using an electrospray deposition technique. Proteins in dots were immobilized by cross-linking in glutaraldehyde vapor. After contact with solutions of isotope-labeled metabolites, the protein microarrays were washed, dried and placed face down onto the surface of a standard B/W video CCD chip with the protective window removed. We show here that such a simple inexpensive CCD detector can be used to quantify distribution of 14C and other radioactive isotopes on microarrays.

Automation↗
PubMed2002

Automated analysis of nanomolar concentrations of phosphate in natural waters with liquid waveguide.

Concentrations of phosphate in natural waters are often below the detection limits of conventional nutrient autoanalyzers, by either gas-segmented continuous-flow analysis orflow injection analysis. A liquid waveguide capillary flow cell has been used to extend the sensitivity of a conventional autoanalyzer for the automated analysis of nanomolar concentrations of phosphate in natural waters. Total reflection of light can be achieved within the liquid core of the flow cell because the refractive index of a cell wall coated with Teflon 1600 is lower than that of water. This property allows the manufacturers to construct long liquid waveguide capillary flow cells in a helical, rather than a linear shape, with compact dimensions. A small sample volume is required because the internal volume of a 2-m long capillary flow cell is only approximately 0.5 cm3. Adaptation of this long flow cell to autoanalyzers significantly enhances the sensitivity of automated colorimetric analysis of phosphate with a molybdenum blue method, allowing for the accurate and precise determination of nanomolar concentrations of phosphate in natural waters. The advantages of this technique are a low detection limit (0.5 nM), a small sample volume (2 mL), high precision (2% at 10 nM levels), and automation for the rapid analysis of a large number of samples.

Automation↗
PubMed2002

Facile method for automated genotyping of single nucleotide polymorphisms by mass spectrometry.

In the future, analysis of single nucleotide polymorphisms (SNPs) should become a powerful tool for many genetic applications in areas such as association studies, pharmacogenetics and traceability in the agro-alimentary sector. A number of technologies have been developed for high-throughput genotyping of SNPs. Here we present the simplified GOOD assay for SNP genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI). The simplified GOOD assay is a single-tube, purification-free, three-step procedure consisting of PCR, primer extension and phosphodiesterase II digestion followed by mass spectrometric analysis. Due to the application of charge-tag technology, no sample purification is required prior to the otherwise very impurity-sensitive MALDI analysis. The use of methylphosphonate containing primers and ddNTPs or alpha-S-ddNTPs together with a novel DNA polymerase derived from Thermotoga maritima for primer extension allow the fluent preparation of negatively charge-tagged, allele-specific products. A key feature of this polymerase is its preference for ddNTPs and alpha-S-ddNTPs over dNTPs. The simplified GOOD assay was run with automatic liquid handling at the lowest manageable volumes, automatic data acquisition and interpretation. We applied this novel procedure to genotyping SNPs of candidate genes for hypertension and cardiovascular disease.

Automation↗