Showing papers by "Daniel B. Oerther published in 1996"

The oligonucleotide probe database.

Abstract: The use of oligonucleotide hybridization probes and PCR primers has become widespread in microbial ecology and environmental microbiology (for reviews, see references 3, 5, 7, 17, and 21), and descriptions of probe applications are abundant in the literature. We have encountered, however, a number of difficulties when relying on the literature for information on probes and primers: (i) probe design, characterization, and application data are scattered throughout the literature and therefore are not easily available; (ii) probe nomenclature is not standardized, making it difficult to recognize a particular probe and evaluate results obtained with that probe; (iii) probes are often designed empirically and used without thorough experimental characterization, making it difficult to interpret experimental results; and (iv) information on the application of individual probes is often not published in detail in the original probe description since the value of some data becomes apparent only as a result of observations made subsequent to publication (e.g., hybridization buffer composition, formamide concentration, membrane supplier and lot number, target group specificity). We designed the Oligonucleotide Probe Database (OPD) to address these concerns. The OPD centralizes information related to the design and use of oligonucleotide probes and PCR primers. The database was originally designed in Microsoft Access Version 2.0 and then converted to Hypertext Transfer Markup Language with PERL scripts. The current data set contains 96 hybridization probes and PCR primers used in microbial ecology and environmental microbiology, published by the authors as well as from direct on-line submissions to OPD. The majority of the probes in the current data set target rRNA, but the database is designed to accommodate probes targeting other gene families. For each probe or primer, the information in the OPD includes design and characterization data important for probe and primer use, including a standardized name, probe sequence, nucleotide position within the target gene, optimal hybridization and wash conditions (or annealing conditions for PCR primers), intended target group, experimentally validated target group specificity, and original citations. Much of the experimental data available in the database were not included in the original publications describing the probes. Standardization of oligonucleotide probe nomenclature. A source of much confusion and frustration during the use of probes or PCR primers designed and characterized in different laboratories has been the absence of a standardized probe nomenclature. Stahl and Amann (18) have previously attempted to address this problem for phylogenetic probes with a nomenclature consisting of three to five letters representing phylogenetic specificity, followed by a number indicating the 59 position on the rRNA complementary to the 39 end of an antisense probe or PCR primer or identical to the 59 end of a sense primer. Limitations to this nomenclature system are apparent when several versions of a probe that have the same target specificity and nucleotide position exist. We modified the nomenclature originally utilized by Stahl and Amann to include multiple probe versions and also to provide additional identifying information. We suggest a method of standardizing the nomenclature for oligonucleotide probes and PCR primers that is both unambiguous and informative. The name for an oligonucleotide probe consists of seven components combined sequentially. These components are discussed below. An example demonstrating construction of probe nomenclature for a small-subunit rRNA-targeted probe is given in parentheses.