MultiPLX 2.1

About the program:

MultiPLX 2.1 is a tool for analyzing PCR primer compatibility and automatically finding optimal multiplexing (grouping) solution. It uses state-of-the-art nearest neighbour DNA binding thermodynamics to estimate possible unwanted pairings between PCR samples. This thermodynamic data is then used to distribute primers into groups that satisfy user defined set of constraints.

Stand-alone MultiPLX binaries, example file and README are available here.

Here is a correct primers file.

For further information please contact:

MultiPLX is a part of services provided by ELIXIR - European research infrastructure for biological information. For other services provided by ELIXIR's Estonian Node visit

How to cite MultiPLX: Kaplinski L, Andreson R, Puurand T, Remm M. MultiPLX: automatic grouping and evaluation of PCR primers. Bioinformatics. 2005 Apr 15;21(8):1701-2. [PubMed] [Full Text]

There have been 609835 submissions to this web site since January 2005.

I Please insert primers file (required for each method!)
(plain .txt file only!)
II Please insert data or/and select method

1. Viewing primer info
This function calculates the following statistics about primers:
1) The melting temperatures of both left and right primer of each pair
2) The strongest binding energies of each type (end-end, end-any, any-any) for each combination of primers in pair (left-left, left-right, right-right). The results can be used as additional estimation of primer quality (unrelated to multiplexing).
Monovalent salts: (mM) Concentration of [Mg+]: (mM)

2. Calculating scores
This function calculates all strongest alignement energies (kcal/mol) for each selected alignement type out of the five possible types (3 types for primer-primer, 2 types for primer-product) between all possible primer pair combinations.
primer-primer end2 primer-primer end1 primer-primer any
primer-product end1 primer-product any  

3. Calculating groups
This function calculates the optimal multiplexing solution of current primers, using strongest binding energies calculated in previous step (or uploaded by user), basic primer parameters (product length, melting temperatures) and optionally additional score file uploaded by user. The resulting file has groups in rows and primer names in columns.
Please insert score file
Choose parameters for grouping
Maximum number of groups
Maximum number of primers in group
Product length max difference (range) in group
Product length min difference (ladder) in group
Maximum primer melting temperature difference in group
Choose grouping stringency
It is possible to use fixed stringency values (high, normal, low) or define custom cutoff values. Fixed stringency values are following:
High: -2.0, -6.0, -6.0, -10.0, -10.0 (kcal/mol)
Normal: -4.0, -8.0, -8.0, -12.0, -12.0 (kcal/mol)
Low: -6.0, -10.0, -10.0, -14.0, -14.0 (kcal/mol)
High Normal Low
dG of primer-primer both 3' ends (kcal/mol)
dG of primer-primer 3' end with any region (kcal/mol)
dG of primer-primer any regions (kcal/mol)
dG of Primer-product 3' end with any region (kcal/mol)
dG of Primer-product any regions (kcal/mol)
Please insert additional score file (Optional)
Maximum allowed score:

4. Verifying groups
This function allows testing existing grouping file (either calculated in previous step or uploaded by user) against score file (again either calculated or uploaded). The resulting file lists all primer pair combinations that break cutoff rules, the types of violated cutoffs and corresponding binding energies.
Please insert score file
Please insert group file
Choose grouping stringency
High Normal Low

University of Tartu, Department of Bioinformatics 2024