AmplifX : Manage, test and design your primers for PCR 

 

Manage, test and design primers for PCR

And do more with version 2!

 

What is AmplifX?

AmplifX is a program dedicated to Polymerase Chain Reaction (PCR) experiments. The three main features are:

Managing primers: keep all the informations for your hundreds or thousands of primers into a single database and retrieve the tubes in the freezer in just a minute! For each primer, AmplifX will calculate some important features: TM, "Quality" (i.e. expected efficiency in PCR reaction), etc. You will be able to add some personnal informations (ID, descriptive name, comments, color coded category, owner, etc.)  You will eventually be able to sort, filter, retrieve, share with your colleagues and of course test them in silico (see below).

Testing primers in in silico PCR: Do not try to remember if you have some primers that can be used for your new project: just test all of your collection in AmplifX and predict PCR efficiency, reaction parameters, amplicon size, etc. 

Design primers: AmplifX can design primers by the way of an intuitive interface: just select the region of interest into you target sequence and let AmplifX choose the best primer sequences or play with intuitive parameters to make more challenging design.  

NEW: AmplifX 2 is now able to design primers and run PCR for dCAPS analysis: it means that the new algorithms allow mismatches in the 3' end of primers that work in real PCR (Stadhouders et al.. J Mol Diagn 2010 and personnal unpublished results)

Legal informations

This program is provided free for the scientific community ”as this”. The author couldn't be responsible for any bad working or lost of data. Users are strongly invited to register themselves with the online form accessible by the help menu and send any bug repport. Registration will allow users to be kept informed for new updates and important informations about this program as soon as they are available.

How to cite AmplifX:

You are asked to cite AmplifX in your publications:

"AmplifX X.X.X [version number] by Nicolas Jullien ; Aix-Marseille Univ, CNRS, INP, Inst Neurophysiopathol, Marseille, France - https://inp.univ-amu.fr/en/amplifx-manage-test-and-design-your-primers-for-pcr"

Download AmplifX

Last Release: AmplifX 2.0.6 

[Oct, 31 2019]

Since the first release of version 2 (as beta release in March 2019), many improvements have been made: so stay up to date!

Version 2 of AmplifX looks like version 1 but is not version 1 ;-)

  • AmplifX has been deeply modified and rewritten for 64 bit support.
  • Many algorithms has been rethought in regard to new insights and real experimental designs in the field of PCR.
  • New functions have been added like CAPS/dCAPS design (for SNP genotyping).
  • The graphic representation has been enriched with GC and complexity content for helping to quickly identify problematic/interesting regions in target sequence/amplicons.
  • Many new features and bug corrections!

Please note that the Version 2 contains a lot of new stuff. It has been intensively tested by the developper and beta testers but you are advised to make backups of your primer lists before using it and send any bug reports as soon as you discover them. (But you shouldn't find too much!) (menu Help > Send feedback)

Mac OS 64 bit application (Apple Disk Image [DMG])

Windows 64 bit application (Setup exe)

Previous last stable release (AmplifX 1.7.0)

[Nov. 21 2013]

 

"Old Mac" (AmplifX Version 1.6.3)

[Nov, 25 2013] These versions allows owners of old Mac to continue to work with AmplifX

 

New Features

Version 2.0.6

Bug fixes of previous v2 beta releases (launched initialy in march 2019):

  • [2.0.6] Fix other bugs! (Amplification with primers that overhangs the target, opening an older AmplifX version regarding of the pref file)
  • [2.0.6] Improves behavior of adding primers to a new unsaved primer list
  • [2.0.5] Fix display of accented characters in feedback forms and alert of new update
  • [2.0.5, 2.0.4] Correction of AmplifX home page URL in the "About AmplifX" window and in the feedback
  • [2.0.3b] Improvement of copy-paste from different binary sources (MacVector, ...) (2.0.3b)
  • [2.0.2b] Fix crash when a primer matches with a "floatting" end on the target (i.e. its start position is before the first nucleotide of the target)
  • [2.0.2b] Fix crash when a shared primer list is closed by the user with write rights and the read-only user is informed he can re open it.
  • [2.0.1b] Improvement of primer suggestion in dCAPS design with very degenerated enzyme sites.
  • [2.0.1b] Add an export button in dCAPS results (to export all results as tab or csv file.)

 

All new features of version 2 in detail: 

  • 64 bit application
  • New functionality for designing CAPS (Konieczny & Ausubel) et dCAPS (Neff et al.) genotyping strategies. (Menu : 'Extra > dCAPS').
  • New match prediction algorithm that is compatible with dCAPS and authorizes mismatches all along the primer (including 3' end). The calculations are based on data from Stadhouders et al. The algorithm starts seeking perfect matches of a sub-sequence (seed) in terminal position (3') of the primer against the whole target sequence and its complementary sequence. For each position where the seed matchs, a "priming score" is calculated based onto data from Stadhouders et al. If the score is above an user-modifiable cutoff, the match is kept. If the preset (see below) uses à number of "seed slide" (N) higher than 1 then the algorithm starts again N-1 times moving the seed from 1 nucleotide to 5' at each iteration. If N is higher than the seed size then it means that one has a chance to get a primer that has at least one mismatch to the target at any position (i.e. dCAPS compatible)
  • New calculation method for match scores (see above) and given on a scale from 0 to 100. Can be considered as chance to work in real PCR (unpublished personal data)
  • New calculation method for the "primer quality" score (0-100 scale).
  • Primers with TM and GC comprised between the min and max cutoffs are now considered all as good. A malus is calculated when these values are out of the range, proportionnaly to the distance and to the tolerance interval (TMmax-TMmin or GCmax-GCmin).
  • For long primers the calculation is done only for the 3' moiety judged relevant (primer with a 5' flanking sequence designed for sequencing or cloning, etc.) The user can mark this by using different font cases (lower or upper) in the sequence. In absence of such case-marked regions the calculation uses only the last 25 nucleotides. On the contrary differences of cases in the last (3') 17 nucleotides are not taken into account.
  • The calculation does not use the previous "polyX" parameter but computes now a lexical complexity score that is more powerful and discriminative.
  • Different "presets" for in silico PCR are provided for allowing balancing sensitivity and speed depending on the user's objectives.
  •          -"Smart" that benefits from all the advantages of the new calculation system and that is fully compatible with dCAPS design (seed of 6 nucleotides and seed sliding 6 times)
  •          -"Classic" that gives almost the same results as AmplifX 1.7  (does not accept mismatches in the last 6 bases of the primer, no seed sliding)
  •          - and  "Fast and furious" for a very fast search with huge primer list of very long target sequences (perfect match of 9 last nucleotides of the primer against the target, no seed sliding)
  • New graphic representation displaying GC content and lexical complexity (both computed on sliding window of 21 nucleotides).
  • New window for restriction enzymes management (data extracted form REbase v812, Roberts RJ et al.) in which the user can filter or select usable/in stock/validated enzymes. 
  • Improvements of the sequence and primer drawings. The zoom can now be at the nucleotide level (displaying in letters). The zoom centers inside the selection
  • Improvement of the zoom management. New keyboard shortcuts ('P': Zoom in, 'M': zoom out, 'O': fit to window, 'N': zoom to nucleotide level)
  • The map can be scrolled with the trackpad
  • Selection of part of the sequence is feasible on the target sequence panel or the graphic map itself 
  • Full screen support
  • Improvement of HiDPI (Retina, etc.) screen support
  • Possibility to modify font sizes (target sequence, primer list, info panel)
  • Possibility to redefine keyboard shortcuts (Pref > Misc.)
  • Possibility to delete from disk a primer file when it's empty.
  • The menus: lower, upper or invert cases, now work onto the edited primer sequence.
  • Fixes a bug of the main window displaying too large (after having changed display resolution beetween two launches of AmplifX, for exemple)
  • Fixes bugs when launching AmplifX by double clicking on a primer list file.
  • ...
  • And many more bug fixes! (Thank you very much to users that report them!)

Known issues:

  • Quick help not translated in english yet
  • Help tags doe not work properly in some situations in the Window version
  • On Windows the display and fluidity of the graphics are not yet optimal!

References:

  • Konieczny, A. and Ausubel, F.M. A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific markers. Plant J. 1993 4, 403-10.
  • Neff MM, Neff JD, Chory J, Pepper AE. DCAPS, a simple technique for the genetic analysis of single nucleotide polymorphisms: Experimental applications in arabidopsis thaliana genetics. The Plant Journal 1998, May;14(3):387-92.
  • Stadhouders R, Pas SD, Anber J, Voermans J, Mes TH, Schutten M. The effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5' nuclease assay. J Mol Diagn 2010, Jan;12(1):109-17.
  • Roberts, R.J., Vincze, T., Posfai, J., Macelis, D.REBASE-a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Res 2015. 43: D298-D299 

See the complete version history

AmplifX : Quick help

Help for version 2 will be available soon!

(Some important features or calculations have changed!!)

  • The target sequence

The main window of AmplifX is organized in two parts: the upper part contains the textual zone, it is organized itself in three panels: "Target", "Primer List" and "Infos".
The panel "target" is the zone in which the target sequence is loaded. one can paste the sequence on which one wants to seek the matching primers. When pasting, AmplifX eliminate all the characters different from : "A, T/U, C, G,". One can thus directly copy from a source containing, for example, spaces, numbers, ... On the other hand AmplifX does not hold account of degenerate nucleotides  (IUPAC: N, S, W...).
AmplifX can open some file formats that are in this current version : ‘DNA Strider', ‘GeneBank', ‘EMBL', ‘GCG', ‘Fasta' and all pure text format that are processed like the paste command (paste only authorized characters)
 It can be useful to mark some regions of special interest by alternating upper and lower cases characters into the sequence (see the paragraph relating to the graphical representation of the target sequence)

  • The Primer list

The panel "Primer List" contains the list of primers and is associated to the " Primers" menu. To import a primer list, use the menu of the same name. It makes it possible to import a file in text format. This one must be formatted as follows: one line by primer with: the primer sequence, its name or ID, its descriptive name or category (There can be others elements which follow but those will not be taken into account). Each item is separated from the others  by a tabulation. This type of file is easily created with Excel or FileMaker, for example. One can also directly enter the primers in AmplifX by creating a blank line with the menu “Add a new primer”.

AmplifX calculates a Quality score, computing some characteristics like TM, GC composition, complexity (polyX and triplet repetitions), 3' stability and self dimers. (the best is 100, the worst is 0)

The “TM” value is computed with three different methods: a simple and very and approximate method: TM=81.5+0.41*GC-675/N, another holding account of the salt concentration in a PCR TM = 81.5 +16.6 X log10([Na+]+[K+])+0.41 x(%GC) - 675/N (with default values: [ Na+]+[K+]=0.05 (50mM)) finally the last one which is the most precise a that time called :"bases stacking method”  : TM=dH/(dS+0.368xNxln[Na+]+ R x ln[Primer]/4) with R=1.987 and the different dH and dS taken in [Santalucia J PNAS 95 pp1460-1465 (1998)]

The list can be sorted by clicking in the headings. It is advised to use data sortable in an alphanumeric way in the column “ID”. For example if you wish to use serial numbers, think of formatting these numbers with a constant number of digits : Pr0001, Pr0002... (the columns Quality and length,calculated by the program are sortable by numerical value) the "descriptive name" column can also be employed to create sub-sections while placing special strings at the beginning of the field, for example one can want to make a category "SYNT" for the primers containing synthetic parts. this will make it possible to quickly find them by simple sorting on this column.

When one double-clicks on a line, either one edit the cell (Sequence, ID, descriptive name) or one selects the whole line. This involves the appearance of a window drawer containing some  details about the quality of the primer and a field “comments” which can be used for any use (name of the owner of the primer, freezer and box, comment on its use...)

The primer list can be saved in a specific format (AmplifX primer list) which is a text file structured in a specific way (it can be opened by any text editor but don't modify it manually). A button in top on the left makes it possible to define a file previously saved as a default file, i.e. automaticaly loaded at AmplifX start up.

  • The informations panel :

The panel infos is self explanory and gives the informations for each clicked graphical element (See below). Its content can be recovered by classic “copy” command.

The PCR chart

The graphic part contains the target sequence drawn as a simple line in two colors relative to positions of upper and lower cases nucleotide characters. At the top and below of this line hybridizing primers appear  as two oriented symbols according to the direction of the  hybridized strand. These symbols are drawn filled or empty property regarding to the strength of the match. (The match cut-off scores can be changed by the preference menu).

The mouse standing onto these symbols let appear the name of the primers and a click above gives, in the panel "infos", the different informations concerning the primer and some hybridization characteristics (Be careful : given TM are those of the primer without holding account of the hybridization quality indeed it does not exist yet method valid for imperfect pairings.)

In the lower part appear the amplified sequences. Their overflight indicates the size of the fragment and the click informs the panel "infos". The click allows also to select directly the portion of the amplified sequence and automaticaly paste the real amplified fragment (i.e. with the primer sequences at the boundaries) into the clipboard (useful to copy-paste in another application).

To change the graphic drawing scale one can use the small buttons "+ ", “-“" and “0” which allow plus zoom, minus zoom or to restore the initial scale. A more practical way to restrict the view to a region of interest is to  drag the mouse while pressing the Shift key. An horizontal scroll bar allow the moving of the viewable portion.

The currently viewable portion of the chart can be printed or exported as standard graphic format by the way of the two dedicated items in the “file” menu

About some menus of special interest :

-The Preferences Menu
It allows the specification of a Primer List file to be open by default.
This one have to be already save with AmplifX. Some constants used for the detection calculations can be modified to allow more sensitive or faster searches. (they are commented into the Pref window)

-The search menu :
A function "Search" in the "Edition" menu makes it possible to quickly locate and select primers whose name or comment fields contain a given string.  The found primers can thus quickly checked for use into the virtual PCR  manually or automatically by the menu "Check found primers”. The search can be done into the target sequence also by clicking the appropriate radio buttion.

-The "Paste complementary strand" menu
 It is useful for the primer design by direct selection in the "Target"field. In which the calculation of quality is carried out “live” as selection change. One can then copy the current selection and paste it either directly or by this menu in a new line of the primer list.

Primer design with AmplifX

Although its first finality is to test already existing primers quickly, AmplifX incorporates since version 1.1 a function of primer design. This function is accessible by the menu "Primers" - > "Design primers". A new window opens which makes it possible to fix some specific parameters to this function, such as the length of the primers wished, the difference of TM between the two ones and the minimum score of quality being used as threshold. This calculation of quality is also dependent on certain parameters which can be changed by the menu " Preferences", such as for example, TM or GC composition.

 One must then fill in a more or less exhaustive way according to what one wishes : the forward and reverse primer searching zones and the length of the product of amplification (if a part of information is not provided the program automatically calculates it with more or less success)
One can choose to search for only one primer by filling one of the two “Use this primer” fields.

Once the search is finished, one can send the primers selected by double-clicking onto the list and clicking on the button "Add to primer list"

Tip : Do not specify a large searching zone for each primer (restricting to 100-200 bp is reasonable), and start by fixing the quality score threshold to a high value ( arround 90). If no primer pairs were found, decrease the constraints then.

If you want to calculate primers in more sophisticated way, I advise you to use a specialized application such as Primer3 [Steve Rozen and Helen J. Skaletsky (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp 365-386]), (a good implementation of this program can be found at this address)

Do not hesitate to help me to  improve AmplifX and this help by sending your comments

They use AmplifX

Last 10 citations (from 167)

  • A Oikari TPS. Use of Early Juvenile Zebrafish Danio Rerio for In-Vivo Assessment of Endocrine Modulation by Xenoestrogens. J Environ Anal Toxicol. 2013;04(01). 
  • Barca A, Vacca F, Vizioli J, Drago F, Vetrugno C, Verri T, et al. Molecular and expression analysis of the Allograft inflammatory factor 1 (AIF-1) in the coelomocytes of the common sea urchin Paracentrotus lividus. Fish Shellfish Immunol. 2017 Dec 1;71:136–43. 
  • Zhang R, Li G, Li X, Shen X, Gao Y, Wang L, et al. A rapid and sensitive recombinase aided amplification assay incorporating competitive internal control to detect Bordetella pertussis using the DNA obtained by boiling. Int J Infect Dis [Internet]. 2019 Sep 1 [cited 2019 Oct 17];86:108–13. Available from: https://www.sciencedirect.com/science/article/pii/S1201971219302735
  • Lee ES, Kim JS, Lee H, Ryu J-Y, Lee H-J, Sonn JK, et al. Auranofin, an Anti-rheumatic Gold Drug, Aggravates the Radiation-Induced Acute Intestinal Injury in Mice. Front Pharmacol [Internet]. 2019 [cited 2019 Oct 17];10:417. Available from: http://www.ncbi.nlm.nih.gov/pubmed/31105565
  • Connors E, Soto-Dávila M, Hossain A, Vasquez I, Gnanagobal H, Santander J. Identification and validation of reliable Aeromonas salmonicida subspecies salmonicida reference genes for differential gene expression analyses. Infect Genet Evol [Internet]. 2019 Sep 1 [cited 2019 Oct 17];73:314–21. Available from: https://www.sciencedirect.com/science/article/pii/S1567134819300899
  • Gaboon NEA, Parveen A, El Beheiry A, Al-Aama JY, Alsaedi MS, Wasif N. A Novel Homozygous Frameshift Mutation in CCN6 Causing Progressive Pseudorheumatoid Dysplasia (PPRD) in a Consanguineous Yemeni Family. Front Pediatr [Internet]. 2019 [cited 2019 Oct 17];7:245. Available from: http://www.ncbi.nlm.nih.gov/pubmed/31294002
  • Ramírez JD, Herrera G, Muskus C, Mendez C, Duque MC, Butcher R. Development of a Digital Droplet Polymerase Chain Reaction (ddPCR) assay to detect Leishmania DNA in samples from Cutaneous Leishmaniasis patients. Int J Infect Dis [Internet]. 2019 Feb 1 [cited 2019 Oct 17];79:1–3. Available from: https://www.sciencedirect.com/science/article/pii/S1201971218345776
  • Apt W, Carrasco D, Fuentealba C, Canals M, Muñoz G, Saavedra M, et al. Chronic Chagas disease: Quantification of Trypanosoma cruzi in peripheral blood and dejections of Triatoma infestans fed by xenodiagnosis in patients with and without cardiopathy. Acta Trop [Internet]. 2019 Dec 1 [cited 2019 Oct 17];200:105167. Available from: https://www.sciencedirect.com/science/article/abs/pii/S0001706X19307508
  • Leonardi P, Murat C, Puliga F, Iotti M, Zambonelli A. Ascoma genotyping and mating type analyses of mycorrhizas and soil mycelia of Tuber borchiiin a truffle orchard established by mycelial inoculated plants. Environ Microbiol [Internet]. 2019 Aug 20 [cited 2019 Oct 17];1462-2920.14777. Available from: https://onlinelibrary.wiley.com/doi/abs/10.1111/1462-2920.14777
  • Pichon M, Labois C, Tardy-Guidollet V, Mallet D, Casalegno J-S, Billaud G, et al. Optimized nested PCR enhances biological diagnosis and phylogenetic analysis of human parvovirus B19 infections. Arch Virol [Internet]. 2019 Nov 10 [cited 2019 Oct 17];164(11):2775–81. Available from: http://link.springer.com/10.1007/s00705-019-04368-w

Complete list of citations (167 articles)

External links

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