How to use:Before you start using Primo Multiplex, the sequence data need to be edited. For beginners, click here to see an example.
Copy the sequences in the example into the input window. On Windows you may need to use Ctrl-C (copy) and Ctrl-V (paste). On Macs you may need to use Apple-C (copy) and Apple-V (paste). Numbers and white spaces will be ignored.
Select the "Multiplex PCR" checkbox, and click on "Go" to start. If the "Multiplex PCR" is unchecked, only one sequence can be used for designing primers.
For the example given above, the job should be finished in 3-5 minutes on most systems. It is not easy to find a set of primers that are consistent in melting temperature, base composition, and don't form primer-primer dimers. Frequently it seems taking forever for the job to finish, that is because the program is attempting to exhaust all possibilities and when it is finished the result is usually "no multiplex primers found."
Frequently the reason is that the regions user selected for 5' and 3' primers don't have normal base compositions, i.e., %GC may be different. Users are suggested to run for individual sequence first to make sure that a number of primers will be predicted for the sequence region and parameters chosen. Run multiplex PCR last using the same or similar parameters. It may be necessary to try several melting temperature and %GC content before finding a multiplex primer set for your sequences. The time taking for designing the primers should be worthwhile since it will reduce the time takes in the multiplex PCR optimization step.
Behind the scene:1. Code for Degenerate Oligos
A A C C G G T T U T M (AC) R (AG) W (AT) S (CG) Y (CT) K (GT) V (ACG) H (ACT) D (AGT) B (CGT) N (ACGT)Any other letters will be ignored, so you can paste in a nucleotide sequences with spaces and numbers.
2. Melting temperature and annealing temperature Melting temperature is the temperature at which 50% of the oligo and its perfect complement are in duplex. PCR annealing temperature a few degree (4-6) lower than the melting temperature is usually used to increase the probability of primer binding. There are two options for calculating the melting temperature. The first uses the simple rule of 2 degree for each A or T and 4 degree for each C or G.
Melting temperature = 4 * Number of G or C + 2 * Number of A or T.
The second "Nearest N" predicts melting temperature using the "Nearest Neighbor" model (Jhon SantaLucia, Proc. Natl. Acad. Sci. Vol. 95, p1460-1465 (1998)). The cation concentration is assumed to be 50 mM and the primer concentration is assumed to be 200 nanomolar. The "Nearest N" is presented because it is more accurate and other formulae can be viewed as approximations of the "Nearest N".
The "Nearest N" formula has a correction for primer concentration. If lower or higher primer concentration is used, the rule-of-thumb is that for each two fold increase or decrease in the primer concentration, the melting temperature should increase or decrease by 1 degree. (Click here to see the relationship between primer concentration and melting temperature.)
For degenerate primers, the effective concentration is lower because of degeneracy. Users don't need to adjust for the lowered effective concentration if using the "Nearest N" formula, Primo has already taken that into account. Users are suggested to select the "Nearest N" formula if template sequences consist of degenerate codes.
To compare melting temperatures calculated using the two formulae, type or copy the primer sequence into the "2nd primer" field, and mouse-click on the text field. The melting temperature will be calculated using the selected formula. Click here to see the melting temperatures calculated using both formulae for some commonly used PCR and sequencing primers.
For degenerate nucleotides, an average is used.
3. Primer-primer dimer
P1: 5'-...GGCGATCG-3' |||||| 3'-GCTAGCGG...-5' : P1or
P1: 5'....AGGGCCC-3' || | 3'-CGCGAAT...-5' : P2The following single-base pair is also not allowed:
P1: 5'-...AGGTCGCG-3' | 3'-CGGATT...5' : P2
4. Random background priming
Black list oligo: 5'-TGCTGCAC-3' Primer 1: 5'-TGCACTACCTGCTGCTGCAC-3' Primer 2: 5'-GCACTACCTGCTGCTGCACC-3' Primer 2: 5'-GCACTACCTGCTGCTGCACCG-3'A primer with mismatch at the last two nucleotides is eliminated because of high probability there will be sequences that match the primer and exonuclease activities of DNA polymerases.
Multiplex PCRTo use the multiplex PCR, select the "Multiplex PCR" checkbox and input multiple sequences in the following format (the same format is used in Primo Pro 3.2 for batch submission):
>seq1 0-100 -100-0 1 ggccgggcgc ggtggctcac gcctgtaatc ccagcacttt gggaggccga ggcgggtgga 61 tcacctgagg tcaggagttc gagaccagcc tggccaacat ggtgaaaccc cgtctctact 121 aaaaatacaa aaattagccg ggcgtggtgg cgggcgcctg taatcccagc tactcgggag 181 gctgaggcag gagaatcgct tgaacccggg aggcggaggt tgcagtgagc cgagatcgcg 241 ccactgcact ccagcctggg caacaagagc gaaactccgt ctcaaaaaaa a >seq2 1 accgcagcgg acagcgccaa gtgaagcctc gcttccctcc cgcggcgacc agggcccgag 61 ccgagagtag cagttgtagc tacccgccca gaaactagac acaatgtgcg acgaagacga 121 gaccaccgcc ctcgtgtgcg acaatggctc cggcctggtg aaagccggct tcgccgggga 181 tgacgcccct agggccgtgt tcccgtccat cgtgggccgc ccccgacacc agggcgtcat 241 ggtcggtatg ggtcagaaag attcctacgt gggcgacgag gctcagagca agagaggtat
Each sequence starts with an info line with a > sign. The description of the sequence may be followed by the optional 5' and 3' range for forward or reverse primers. The three fields are separated by "tab."
If the range value starts with a minus sign, then the counting will be from the 3'-end of the sequence. If both ranges are missing in the info line, the input value from the Primo interface will be used. If only one range is present in the info line, that value will be used for designing forward or reverse primer only. It will be ignored for selecting pairs of forward and reverse primers.
Species available in the stand-alone version
Contact us if a species you are interested in is not on this list, we might be able to help.