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Primo Multiplex 3.2 Help: Multiplex PCR Primer Design
About Primo Multiplex 3.2: Online
Multiplex PCR amplifies multiple DNA fragments in one PCR reaction. One frequent problem is that although individual PCR works well, when all in one tube, they don't. One of the reasons is that primers form primer-primer dimers. As the number of primers increase in a multiplex PCR, the possibility of
dimers increases exponentially. Subtle differences in
primer melting temperature and base composition may also results in bigger
differences in amplification efficiency when primers are competing
for enzyme and template.
Primo Multiplex searches for sets of primers that are consistent in characteristics and don't
form dimers for any combination of the primers.
For normal PCR primer design, please use
Primo Pro 3.2. Primo Pro 3.2 introduces an option for reducing PCR noise by lowering the probability of random
primering on non-target DNA sequences. This option is kept in
Primo Multiplex. For example the primer TGCACTACCTGCTGCTGCAC for
the p53 gene looks perfectly OK because it meets most criteria we commonly use for designing
PCR primers. This primer may have high background amplification because the 3’-prime 8 nucleotides
appears in 4800 unique genes in human transcriptome. Primo Pro analyzed in advance
transcriptomes of different species to mark sequences that are over-represented, thus allow users to
select primers with few over-represented sequences at the 3’-end. As a result of reduced random
priming, we expect improved PCR amplification efficiency and cleaner PCR products, especially for
RT-PCR reactions.
Primo Pro 3.2 also introduces a batch mode option for high throughput
PCR primer design. By selecting the batch mode, users can input
multiple sequences and design PCR primers for multiple sequences. Batch
mode is adapted for multiplex primer design in Primo Multiplex.
A lot of calculations have been done in advance for Primo Pro.
That is why Primo Pro runs amazingly fast in your web browser.
But to take full advantage of Primo Pro, you would need to install it on your computer.
For example, the online Primo does not allow you to chose a species other than human
because limitations in passing large number of data on the web. Note it searched
only transcribed sequences and each species has a unique pattern of
over-represented sequences. Unlike proteomes of closely related species,
transcriptomes of close relatives usually don't share over-represented
sequences, probably because untranscribed regions are less conserved
and/or many species have young transposons.
The stand-alone version of Primo Pro 3.2 and Primo Multiplex 3.2 currently has
transcriptome data for 11 species. More species will be added in the near future.
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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 OligosA 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
If you select to "Check primer-primer dimers", Primo Pro 3.2 eliminates primers
that might form primer-primer dimers such as the following:
P1: 5'-...GGCGATCG-3'
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3'-GCTAGCGG...-5' : P1 or
P1: 5'....AGGGCCC-3'
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3'-CGCGAAT...-5' : P2
The following single-base pair is also not allowed:
P1: 5'-...AGGTCGCG-3'
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3'-CGGATT...5' : P2
4. Random background priming
Primo Pro has blacklisted a large number of over-represented sequences of 8 nucleotides by
analyzing transcriptome data from a number of species. Note there will be
a unique list for each species. A primer candidate will be compared to the
blacklist for matches in the last 10 nucleotides. Any one with a match will be
eliminated from the candidate pool. The following three primers will be
eliminated:
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 PCR
To 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
| Species Name | Common Name |
| Arabidopsis thaliana | Arabidopsis |
| Zea mays | Corn |
| Bos taurus | Cow |
| Drosophila melanogaster | Drosophila |
| Xenopus laevis | Frog |
| Homo sapiens | Human |
| Mus musculus | Mouse |
| Rattus norvegicus | Rat |
| Oryza sativa | Rice |
| Danio rerio | Zebrafish |
Contact us if a species you are interested in is not on this list, we might be able to help.