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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.

Browser requirements:

Primo Pro 3.2 Online and Primo Multiplex 3.2 Online runs on the following Java-enabled browser:
  • PC: Internet Explorer 5.5 or higher and Netscape 4.08 or higher.
  • Mac (OS 9.*): Internet Explorer 5.0
  • Mac (OSX10): Early version of OSX10 IE5.1 has a bug. Copy and paste will kill the browser. The bug should have been fixed in later versions of Mac IE.

    If you use one of the above browser and you can't run Primo Pro 3.2, please make sure Java is enabled in your browser. For Internet Explorer, go to Tools/Internet Options, click on Security Settings, scroll down to find Microsoft VM, deselect "Disable Java". Mac OSX 10 IE5.1 has a bug, it does not allow copy/paste into a Java text field, thus you will not be able to import a new sequence. Attempting copy/paste may kill your browser. Stand-alone versions don't require IE to run, thus don't have this problem.
  • 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
    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'
                ||||||
             3'-GCTAGCGG...-5' : P1 
    or
    P1: 5'....AGGGCCC-3'
                 || |
              3'-CGCGAAT...-5' : P2 
    The following single-base pair is also not allowed:
    P1: 5'-...AGGTCGCG-3'
                     |
                  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.

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