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Primo Degenerate 3.2 Help: Degenerate PCR Primer Design
About Primo Degenerate 3.2: Online
Degenerate PCR is frequently used to clone unknown genes in a family. Primo Degenerate 3.2 designs PCR primers based on a single peptide sequence or multiple alignments of proteins or nucleotides. For degenerate primers, the probability of binding to the target is proportional to the effective concentration of the specific primer (i.e., inversely to the fold of degeneracy). Most programs and models (e.g., 2 °C for each A and T, 4 °C for each C and G) did not take this into account thus will predict a much too high melting temperature. By using the Nearest Neighbor Tm model, Primo automatically adjusts Tm for degenerate primers, resulting in better primer design.
How to calculate melting temperature for degenerate 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:
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 "Degenerate PCR" checkbox, and appropriate options for the input sequence (Single protein, Multiple proteins, or multiple nucleotides) and click on "Go" to start.
The Nearest Neighbor Tm formula is suggested for designing degenerate primers since it will automatically correct for the effects of primer degeneracy. Users may select the AT=2 CG=4 formula to disable this feature. As explained in how to calculate melting temperature for degenerate primers, degeneracy results in lowered melting temperature.
Users are suggested to start with low melting temperature, any GC% and 3'-end. Next raise the stringency to improve the quality of primers.
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 missmatch 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.