#ONLINE PRIMER DESIGN TOOL PDF#
#ONLINE PRIMER DESIGN TOOL SOFTWARE#

favour a small amplicon size for reproducible and robust assays.

favour intron spanning amplicons to remove false signals from contaminating genomic DNA.

favour a unique assay without cross hybridizations to other areas of the genome ( in silico PCR).

search for gene family members and splice variants.

search genome to ensure primer uniqueness.

the human, mouse and rat design relies on the 90 probes of the respective organism specific UPL sets, where as the remaining are based on the complete 165 UPL probes.

search input sequence for Universal ProbeLibrary target sites avoiding known human SNP's (only for Ensembl sequences).Finally, ProbeFinder applies a correction to identify intron sites with the consensus 5' and 3' splice sites (GT and AG). If only one exon can be found in the target gene sequence, the sequence will not be considered an intron unless its identity is 100% certain, the exon is at least 100 nt long, and the intron is at least 200 nucleotides long. the intron must be at least 30 nucleotides long.Ī sequence is considered an intron if it has two flanking exons (as defined by BLAST matches).the exon must be at least 40 nucleotides long, and.the identity must be at least 95% certain,.ProbeFinder uses the following criteria when predicting introns: in-house prediction algorithm based on BLAST.Introns are identified by one of the following methods: The following databases are available to ProbeFinder: h_sap_gene, h_sap_exon, h_sap_refseq, h_sap_embl, h_sap_genome. ProbeFinder performs a number of steps to select the most optimal real-time PCR probe from the Universal ProbeLibrary in combination with a set of PCR primers. When gene names or keywords are entered, ProbeFinder provides results from a number of databases containing your keyword, to help you select the gene ID or nucleotide sequence. Currently acceptable entry formats include RefSeq, GenBank/EMBL and Ensembl sequence IDs. Target information can be entered either by typing a gene accession number, gene name or keyword, or the target nucleotide sequence. The assay design process is startet by selecting the appropriate organism and entering target information in the depicted input windows. The default settings were choosen to give best PCR results with Universal ProbeLibrary probes without any further optimization of assay conditions, as described in the product information.

Preferably, the selected PCR amplicon will span an exon-exon junction and be 60 - 150 bp long.Įxperienced Primer3 users can modify these settings before they start assay design.

#ONLINE PRIMER DESIGN TOOL SOFTWARE#

ProbeFinder assay design software is based on Primer3 software using optimized settings as default, e.g., the melting temperature for both primers is set at +59 to +61☌ and the primer length is set at 18 - 27 nucleotides. Together, the probe and PCR primers constitute a specific real-time PCR assay for a given target. No., gene name, or sequence), the software designs optimal real-time PCR assays by combining a suitable Universal ProbeLibrary probe with a target specific set of PCR primer. Based on the user-defined target information (Gene Acc. ProbeFinder is a web-based software tool that is used in combination with the Universal ProbeLibrary probes. Detailed information on all assays are displayed and the most suitable can be chosen. You can also use functions such as selecting a multiplex assay with a reference gene, designing up to 10 different assays at a time, or differentiating between members of a gene family or different splice variants. The software allows you to simply design an intron-spanning assay by entering gene name, accession number, or sequence.

#ONLINE PRIMER DESIGN TOOL PDF#

Results can be downloaded as pdf or text report. In addition the reference gene assay option for multiplexing is depicted. Assay information contains, probe number, primer and amplicon sequence. ProbeFinder always displays the best assay according to ranking criteria. Otherwise, simply deselect 'intron spanning assay'. Known intron-exon boundaries can be marked with square brackets in order to design an intron-spanning assay. If your favorite organism is not in the list, you can still design an assay select 'other organism', and paste in an appropriate part of the sequence of your gene of interest. Universal ProbeLibrary Assay Design Center

Online primer design tool