PubMed · 60665
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E J Stokes. 1976-09-11. Letter: Automated test for bacteriuria.. https://doi.org/10.1016/s0140-6736(76)91838-9
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HLA class II typing by DNA amplification and hybridization with allele-specific oligonucleotides has led to an improvement of the overall typing accuracy. In an attempt to apply this procedure to a large-scale analysis, we sought to implement an automation system using the Biomek 1,000 robotic work-station. This system allows a rapid preparation of many consecutive samples and avoids the risk of human errors. It is now routinely used in several laboratories.
We report the design, chemical synthesis, and structural and functional characterization of a novel reagent for protein sequence analysis by the Edman degradation, yielding amino acid derivatives rapidly detectable at high sensitivity by ion-evaporation mass spectrometry. We demonstrate that the reagent 3-[4'(ethylene-N,N,N-trimethylamino)phenyl]-2-isothiocyanate is chemically stable and shows coupling and cyclization/cleavage yields comparable to phenylisothiocyanate, the standard reagent in chemical sequence analysis, under conditions typically encountered in manual or automated sequence analysis. Amino acid derivatives generated with this reagent were detectable by ion-evaporation mass spectrometry at the subfemtomole sensitivity level at a pace of one sample per minute. Furthermore, derivatives were identified by their mass, thus permitting the rapid and highly sensitive determination of the molecular nature of modified amino acids. Derivatives of amino acids with acidic, basic, polar, or hydrophobic side chains were reproducibly detectable at comparable sensitivities. The polar nature of the reagent required covalent immobilization of polypeptides prior to automated sequence analysis. This reagent, used in automated sequence analysis, has the potential for overcoming the limitations in sensitivity, speed, and the ability to characterize modified amino acid residues inherent in the chemical sequencing methods that are currently used.
The methods used for the isolation and identification of obligate anaerobes often appear to be exactly the same as those used 20 years ago. However, careful evaluation of the methodology available reveals many improvements in technology. More importantly, the use of computerized instrumentation and new, non-growth-dependent methods for characterization suggest that in the next few years even greater changes will occur in systems for identification of this group of organisms.