OligoCheck Help

The ability of nucleic acid primer sets and probes to bind exclusively to the correct nucleic acid sequence is fundamental to the accurate targeting of specific phylogenetic groups.  Such specificity depends largely on the uniqueness of the region of nucleic acid sequence these oligonucleotides target.  The more unique the region, the more robust will the primer set or probe be in annealing only to the intended target, even if  variations in annealing temperature and other biochemically relevant parameters result in the oligonucleotides binding successfully with targets with one or more base differences.  Thus, a robust primer set or probe can be viewed as one that anneals exclusively to its intended target even when one or more mismatches are allowed.

OligoCheck is designed to assess this robustness, by assessing a primer set or probe's phylogenetic range as the number of base-mismatches with potential target sequence increases.  The user supplies a primer set or probe to check, along with a database of nucleic acid sequences.  OligoCheck checks the primer set or probe against each relevant record in the database and notes whether matches occur with an increasing number of base mismatches allowed.  The resulting analysis is displayed in a graphical form alongside the sequence-record data.

Quick Start Guide

Main Window

The primer set or probe to be checked is entered in this window, either manually or by loading a pre-prepared oligo file via the File menu.  Database(s) to be checked against are then loaded by selecting the 'Load Database' button.  Once this has been done the analysis can take place by clicking on the 'Do Analysis' button.  Once analysis is complete, a result summary is displayed in the main text box of this window.  Clicking on 'View Results' enables a full breakdown on the results to be viewed.  The options pull-down menu enables the user to alter certain parameters associated with the analysis.

Load Database

OligoCheck considers primer sets or probes against one or more databases supplied by the user.  Use this dialog box to load the required database(s) into the program.  For analyses involving oligonucleotides that target the 16S rRNA gene, the user may wish to load the RDP aligned database by clicking on 'Load RDP Database' button.  All other databases are loaded by clicking on 'Load Other Database(s)'.  Once all required databases have been loaded and any records not required de-selected, click on 'Process Database(s)' afterwhich this dialog box can be closed.

Results Window

The analysis completed, this dialog box displays the results of the analysis.  During the analysis (initiated by pressing 'Do Analysis' within the main window), the supplied primer set or probe will have been checked against each database sequence.  Matches between oligonucleotide(s) and database-sequence, with increasing number of base-mismatches allowed will have been noted.  The resulting output of this analysis is presented within the results window as a graph.

Tutorial

• Example 1 - checking a primer set.
• Example 2 - checking a probe.