Avoiding Cross Homology- For specific Oligo Design
Oligos binding to regions of the sequence other that the target leads to mispriming and false positive signals. To avoid this, Beacon Designer™ interprets the BLAST search results and avoids the regions that have significant homologies with the database sequence. You can directly BLAST search from Beacon Designer™ and do not need to go to the NCBI BLAST site. It is recommended that you run a BLAST search previous to performing a primer-probe search to increase their efficiency. You can set the genome category against which you want to BLAST search, E value of the matches (by default set to 10), whether or not you want to apply filters to the query sequence (to mask repeats and low complexity regions) and the format of the ouput report.
Verifying Specificity of Design
You can BLAST search the primers/probes or the amplicon against a genome to verify the specificity of the design. In the report generated, you can see the Score and E value of the significant alighments that are reported.
Local BLAST and Desktop BLAST
For researchers working with confidential data or who do not have a fast internet connection, Beacon Designer™ can interpret the BLAST results from a local database. You could install a local custom database using the WWW Standalone BLAST facility of the NCBI. This database has to be installed on a server machine with Unix/Mac OS X. You can also set up a local daytabse of sequences/genome on your own machine. This option is present as "Destop BLAST". Desktop BLAST is easy to configure and use. Just download and save the sequence files in Genbank format (starting with a ">" sign) with a .txt or .fa extension.
Avoiding Template Structures- Increasing Primer Efficiency
Primer extension might be hindered if during the PCR reaction, the template (sequence) folds upon itself and forms loops. This happens when the complementary adjacent base pairs in a region form bonds. To avoid all such regions, where the template might fold upon itself, Beacon Designer™ does a template structure search. The current sequence range that can be check for secondary structures is 1200 bp. You can also specify the temprature at which you want to fold the template. The results are automatically interpreted and the regions are avoided when designing primers.
Automated Doesn't Means No Flexibility
Beacon Designer™ saves time by automating the real time PCR assay design process. Yet, you can finely control the design and aks the program to give your choices. Some examples:
You can restrict the assay design in a particular region of you sequence say near the 3' end or the 5' end or even specify a range on the sequence where you which to design an assay.
You can specify the primer Tm or Ta.
You can get upto 50 alternate primers/probes/primer probe sets for your assay to choose from.
You can set maximum dG values for primer parameters such as hairpin, self dimer cross dimer and 3' end stability. You can set the maximum length of runs and repeats and GC clamp at the 3' end of the primers.
You can specify maximum primer Tm mismatch and cross dimer values.