No, it cannot be downloaded. PrimerChecker℠ is a Web interface platform of free access through the internet and any use or related material to it are provided as a courtesy, free of charge, and are intended for informational and educational purposes only. See the PrimerChecker℠ disclaimer under the tab 'About'.

There is no App version of PrimerChecker℠. However, it displays and performs reasonably well in most smartphones.

Yes, it is!

Yes. Use the function "Load file..." if handling a large number of oligos. This function will facilitate your handling of large data sets faster than type directly the info into the table. The "Load file..." function allows you to load your own data-set of primer parameters using Excel files (.xlsx, .xls file extensions) or text files with tab separated values (.tsv or .txt), use the template file available from the "Download template" button. Additional details about this function and required file format are described in the section File Format of Extended Help. You can plot single primers or pairs, then decide which combination of primers is optimal.

All parameters are important. PrimerChecker℠ delivers holistic plots of thermodynamic parameters to facilitate decision making by the operator about the quality of oligos that were designed or are intended for use for PCR amplification of DNA. The user is encouraged to develop a holistic perspective to ponder equally all thermodynamic parameters presented plotted against optimal, good or suboptimal ranges for rapid visualization and analysis.

Therefore, if seeking optimal primer performance and maximum sensitivity, ΔTm must be 0 or at the minimum possible to assure both primers will anneal simultaneously favoring the dynamic and efficiency of the PCR reaction.

Equally important is the tendency of primers to form secondary structure at the 3'. The 3' terminus is the most important region of the primer, a mismatch or secondary structure at the 3' terminus (rattle snake structure) will interfere or deter the polymerase to move forward with the synthesis. The 3' value should be 0 for maximum performance, but this is not always possible. The more it is compromised the less the sensitivity. Is sensitivity critical for you (i.e. gene expression/virology)? For some it is not. In like manner, is the tendency of the whole primer to form secondary structure as it is expressed by the ANY score calculated by Primer3 and/or Primer-Blast relevant? This parameter value should be as low as possible. ΔG is enormously critical because the change in the free energy of the system occurring during a reaction measures the balance between the two driving forces that determine whether a reaction is spontaneous. Any reaction for which ΔG is negative should be favorable, or spontaneous (which is advantageous). Designing primers with negative ΔG only is frequently difficult, and ultimately will

Not a problem. You can actually calculate these using your primer sequences (5'-3'). Go to the NCBI-NIH Primer-Blast and access your two primer sequences at each of the 'Primer Parameter' boxes. Scroll down to 'Primer Pair Specificity Checking Parameters' and select the database that goes with your research-work (we mostly use 'nr') then type the scientific name of the organism you are working with. You will retrieve all parameter values except the energy plot ΔG. You can retrieve the energy plot ΔG using the IDT's OligoAnalizer 3.1. After submitting your sequence click on 'Hairpin'. You will be able to see the ΔG in plot from the top of the generated mFold energy dot plot, seen as a little square at the right under 'General Information'.

You can also consider visiting another web application called mFold to calculate the ΔG in plot. To do so, enter the sequence of the primer to be analyzed in the box below. Scroll down to the DNA sequence box, select 'linear'. In the box 'Folding temperature' select the PCR Tm of the reaction your primers were designed for or will be working at. Commonly around 60°C. At the box 'Ionic conditions' tick on 'mM' and type in the [Na⁺] box 10 (it will depend on your reaction) and write 1.2 -2.7 in the box [Mg⁺⁺] or the Mg⁺⁺ depending on the PCR mix of your assay or commercial pre-mix in use. Consider you might want to use values of [Na⁺] until 50 mM and [Mg⁺⁺] for 3-6 mM depending on the conditions of the assay you are designing. Once you get the output click on 'jpg' type of file at the 'energy dot plot'. Your ΔG is at the top of the plot.

PrimerChecker℠ does not 'analyze'. PrimerChecker℠ generates primer quality plots to facilitate a visual interpretation and analysis by the operator.

Back to your question... Sure! How do you know the primers being used wouldn't perform better? You can generate plots of 'believed good' primers to confirm they are optimal or good indeed, or alternatively deciding the primers may be improved or redesigned if found to be suboptimal not as good as thought. Frequently, just by adding or deleting one or few bases primers may improve considerably. I mean 'moving' the primer sequence one or few bases upstream or downstream the template sequence. If doing so, replot with PrimerChecker℠ using new parameter values for decision making.

Yes. The missing data will be plotted as suboptimal and the missing parameter will be seen in the lower suboptimal segment of the plot.

This is a good question and a tricky situation for PrimerChecker℠, but still doable... First consider degenerate primers are actually a primer mixture of all designed variants. You can obtain the thermodynamic values of your degenerate primer sequence with the IDT's OligoAnalizer 3.1. After submitting your sequence click on 'Analyze' and you will retrieve the Melting Temperature (Tm) range (Min, Mean, Max), similarly click on 'Hairpin'. You will be able to retrieve the ΔG in a plot from the top of the mFold energy dot plot file generated for your analysis (seen as a little square) at the right under 'General Information'. You can also consider the ΔG (Kcal/ Mole^-1) for maximum and minimum of the calculated structures.

Your task will be generating plots for your degenerate primers with min., mean and max. Tm data.

Not at the moment. You can submit thermodynamic data for an internal qPCR probe, but consider that the plot may not be informative within the context of PCR. The reason is that the thermodynamic parameters of internal probes differ from those applicable to the respective primers. For example, the Tm of internal hybridization probes is 8-10°C higher than the primer's Tm which ensures the probe is annealed in place before the primers anneal and the polymerase synthesis take off. If bindings do not occur in this sequence, the assay output is at risk of being a false negative. Moreover, the ANY score calculated by Primer3 and the secondary structure relative to the 3' terminus are more relaxed for probes and not that strict as for primers.

The good news is that this type of plot will be offered in the future. It depends of funding availability.

Yes you can. For a more complete analysis consider plotting the primers before and after adding the 5' non-complementary nucleotide extension. This way you will get the extent of improvement achieved after modifying the primer.

No. However, PrimerChecker℠ will plot the thermodynamic values of the primers that can or will be labeled.

If the core primer sequences cannot be altered, PrimerChecker℠ plots will inform the operator which thermodynamic parameter needs work. If the primers' Tms differ from each other by 5°C then adding 5' non complementary G/C nucleotides not complementary to the template, to the primer with lower Tm will raise this primer Tm to equilibrate them (see Arif & Ochoa-Corona Mol Biotechnol (2013) 55:17-26). The nucleotide addition will be empirical until equilibrating both primers Tm to the same or similar Tm. You can check the targeted template sequence and adding extra bases as well to raise the Tm, but check the new secondary structure of the primer because it may change.

Sure! Please e-mail us your data. It will allow us to further analyze and eventually improve the selected ranges of each of the plotted thermodynamic parameters.

Mg⁺⁺ and Na⁺ concentrations are still relevant conditions to test during the in vitro optimization of the reaction. PrimerChecker℠ is a bioinformatics tool for graphic display of thermodynamics parameters of oligonucleotide primers for DNA amplification and prediction of their performance in silico. What this mean in practice is that once you have designed primers with any software of your choice (but preferable Primer3) you can generate PrimerChecker℠ quality plots with the thermodynamic parameters obtained during primer design to visualize in a holistic way the performance during a PCR reaction and deciding if good enough to continue with the in vitro (wet) stage.

Considering the ranges selected for qualitatively plotting each of the thermodynamic parameters I would say End Point PCR is the primary application for PrimerChecker℠.

No. End Point PCR is the primary application for PrimerChecker℠. However, this is a tricky scenario and PrimerChecker℠ can give some light to your problem. There are similarities between primers designed for End Point PCR and HDA. Regarding LAMP you can plot the most external primers F3 & B3 considering they are essential for LAMP to take off. For both techniques HDA and LAMP (F3 & B3) the reaction temperature is circa 65°C and for PrimerChecker℠ the optimal Tm ranges varies >52<65°C. For RPA primers are longer and the temperature of the reaction is low (37°C). In both cases RPA and LAMP denaturation of primers is not heat driven but worked out enzymatically. The good news is that primer plots adjusted to HDA, RPD and LAMP parameters will be offered in the future. It depends of funding availability.

Yes.

Yes.

Yes.

Sure! Please do not forget to cite PrimerChecker℠.