Roche Qpcr Primer Design Tool

Universal ProbeLibrary System Assay Design

Read in this article:

Overview

Other Organisms

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. Known intron-exon boundaries can be marked with square brackets in order to design an intron-spanning assay. Otherwise, simply deselect "intron spanning assay".

Display of Results

ProbeFinder always displays the best assay according to ranking criteria. Assay information contains, probe number, primer and amplicon sequence. In addition the reference gene assay option for multiplexing is depicted. Results can be downloaded as pdf or text report.

Display of results in the Assay Design Center

Additional Functions

The software allows you to simply design an intron-spanning assay by entering gene name, accession number, or sequence. 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. Detailed information on all assays are displayed and the most suitable can be chosen.

ProbeFinder Assay Design Software

ProbeFinder is a web-based software tool that is used in combination with the Universal ProbeLibrary probes. Based on the user-defined target information (Gene Acc. 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. Together, the probe and PCR primers constitute a specific real-time PCR assay for a given target.

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°C and the primer length is set at 18 - 27 nucleotides. Preferably, the selected PCR amplicon will span an exon-exon junction and be 60 - 150 bp long.
Experienced Primer3 users can modify these settings before they start assay design. 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.

Input Formats for Target Specification

The assay design process is startet by selecting the appropriate organism and entering target information in the depicted input windows.
Target information can be entered either by typing a gene accession number, gene name or keyword, or the target nucleotide sequence. Currently acceptable entry formats include RefSeq, GenBank/EMBL and Ensembl sequence IDs. 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.

Steps of the Assay Design Process

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. The following databases are available to ProbeFinder: h_sap_gene, h_sap_exon, h_sap_refseq, h_sap_embl, h_sap_genome. Database updates are done regularly.

1	Locate exon-exon junctions	Introns are identified by one of the following methods: look-up in Ensembl (if available) in-house prediction algorithm based on BLAST user annotated in the input sequence ProbeFinder uses the following criteria when predicting introns: the identity must be at least 95% certain, the exon must be at least 40 nucleotides long, and the intron must be at least 30 nucleotides long. A sequence is considered an intron if it has two flanking exons (as defined by BLAST matches). 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. Finally, ProbeFinder applies a correction to identify intron sites with the consensus 5' and 3' splice sites (GT and AG).
2	Find appropriate UPL probe	search input sequence for Universal ProbeLibrary target sites avoiding known human SNP's (only for Ensembl sequences). 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.
3	Design PCR primer for each target site	search genome to ensure primer uniqueness search for gene family members and splice variants performin silico PCR
4	Rank the available assays to	favour a unique assay without cross hybridizations to other areas of the genome (in silico PCR) favour intron spanning amplicons to remove false signals from contaminating genomic DNA favour a small amplicon size for reproducible and robust assays best multiplex combination with selected reference gene
5	Display results	ProbeFinder always displays the best assay according to the above described ranking criteria. Assay information contains probe number, primer and amplicon sequence. In addition the "Multiplex PCR with Reference Gene" option is depicted. Results can be downloaded as pdf or text report. in the "Transcript Overview" primer and probe positions on the complete transcript are shown. When a sequence identifyer from the GenBank/EMBL (e.g.,ENST00000217133.1) was entered, SNPs of the whole transcript are displayed and details can be seen with the mouse over function. the "Detailed View" shows the aplicon area enlarged with detailed information about amplicon length and SNP positions. when the "More Assays" option is selected, all possible assays for your gene of interest (with or without reference gene assays) are displayed in detail so that you can select the best assay for your particular experiment.

In silico PCR - increases the success of your designs

Thein silico PCR feature automatically performs virtual assays mimicing a real-time PCR assay.
All primer pairs designed by ProbeFinder (using Primer3) are checked by an in-house developedin silico PCR algorithm. The algorithm searches the relevant genome and transcriptome for possible mis-priming sites for either of the two PCR primers. If any of the identified mis-priming sites are positioned in the genome or the transcriptome in a way that could potentially give rise to an unintended amplicon, the assay is downgraded in the list of available assays and flagged as having failed thein silico PCR check.

Thein silico PCR function minimizes:

risk of false assay signals from genomic DNA
risk of false assay signals from unrelated transcripts generated by splice variants or homologous genes/gene family members
detection of pseudogenes
targeting of genes with introns that are too short for effective intron-spanning assays

Multiplex assays with human, mouse or rat reference genes
When the option "Design multiplex PCR with reference gene" is selected, ProbeFinder will conduct assay design for your gene of interest, while at the same time subjecting each of these designs to anin silico test to validate its ability to be multiplexed with the UPL reference gene assays you selected. In the event no matching assays are found for your selection, ProbeFinder will display the best available combination with one of the reference gene assays available for the relevant organism. Thein silico PCR for multiplex assays takes the following parameter into consideration:

primer-primer interactions (to avoid formation of primer-dimers)
primer-probe interactions (to prevent probe and primer competing for binding sites or binding to each other)
probe-probe interactions (to avoid probe-probe binding)
probe-amplicon interactions (to prevent the probe from incorrectly generating signal on the amplicon).
in silico PCR with all 4 primers (to prevent amplification of undesired cDNA fragments)

Entering Multiple Sequences

Batch processing - design assays for up to 10 different transcripts at once.
Rather than having to design assays on a gene-by-gene basis, "Batch processing" allows you to simultaneously generate assays for up to ten targets at a time.
"Batch processing" is the default setting for ProbeFinder software. ProbeFinder will design an assay for each of the genes as though it had been entered individually. In addition, the software includes two optional features that allow you to analyze these results in two different ways, by selecting "Differentiating Assays" or "Common Assays".

"Differentiating Assays" - design assays to discriminate between closely related sequences, gene families, or splice variants.
Once ProbeFinder has displayed the results for the submitted batch of transcripts or sequences, you can further refine the assays. The "Differentiating Assays" feature is particularly useful when you want to target a particular splice variant of your gene. When you select this function, ProbeFinder applies strict criteria to identify those assays that are specific to each of the submitted gene family members or splice variants. In this way, ProbeFinder does its utmost to design an assay that is truly specific for the selected transcript or gene. If a unique design cannot be chosen, ProbeFinder will not generate a solution.

"Common Assay" - design a single assay for related genes or splice variant families.
The "Common Assay" feature tries to design one assay that can be used to assay more than one gene in a family or a set of splice variants. It is particularly useful when you want to assay the total product of a gene,i.e. all splice variants, rather than one particular splice variant.

Other Organisms

When your organism of interest is not available in the drop down menue on the Assay Design Center, you can still use UPL and the ProbeFinder software to design assays. The Universal ProbeLibrary probes can be used to analyze any organism, and assays can be designed for any sequence from any organism (or any sequence from a non-natural source) provided that the sequence contains a probe binding site and corresponding primer sites.

To design an assay for such a sequence select the button "Other Organisms" and paste the sequence into the "sequence"-field.
ProbeFinder will automatically search for an assay amongst all 165 Universal ProbeLibrary probes.
Since ProbeFinder cannot use the genomic database for your selected organism, ProbeFinder cannot design intron-spanning assays, unless the position of the exon-intron-junctions in your sequence are marked with open brackets [ ] in the input window.
In case, the junctions are not know, it is necessary to deselect the "Automatically select an intron spanning assay" button to receive a list of available assays for the requested sequence.

In order to have a complete set of all 165 probes available in your freezer, you can order the Universal ProbeLibrary Set, Human, which contains probes #1-90 and the Universal ProbeLibrary Extension Set, Probes #91-165.

ProbeFinder Quick Reference Guide
The ProbeFinder software allows you to perform a fast and easy design of real-time PCR assays for your targets of choice.
Manual (PDF, 718 KB)